Microfluidic particle-analysis systems

ABSTRACT

The invention provides systems, including apparatus, methods, and kits, for the microfluidic manipulation and/or detection of particles, such as cells and/or beads. The invention provides systems, including apparatus, methods, and kits, for the microfluidic manipulation and/or analysis of particles, such as cells, viruses, organelles, beads, and/or vesicles. The invention also provides microfluidic mechanisms for carrying out these manipulations and analyses. These mechanisms may enable controlled input, movement/positioning, retention/localization, treatment, measurement, release, and/or output of particles. Furthermore, these mechanisms may be combined in any suitable order and/or employed for any suitable number of times within a system. Accordingly, these combinations may allow particles to be sorted, cultured, mixed, treated, and/or assayed, among others, as single particles, mixed groups of particles, arrays of particles, heterogeneous particle sets, and/or homogeneous particle sets, among others, in series and/or in parallel. In addition, these combinations may enable microfluidic systems to be reused. Furthermore, these combinations may allow the response of particles to treatment to be measured on a shorter time scale than was previously possible. Therefore, systems of the invention may allow a broad range of cell and particle assays, such as drug screens, cell characterizations, research studies, and/or clinical analyses, among others, to be scaled down to microfluidic size. Such scaled-down assays may use less sample and reagent, may be less labor intensive, and/or may be more informative than comparable macrofluidic assays.

CLAIM OF PRIORITY

This application claims the benefit of priority under 35 U.S.C. §119(e)to provisional applications Ser. No. 60/369,538, filed Apr. 1, 2002 and60/378,464, filed May 6, 2002, both of which are hereby incorporated byreference in their entirety for all purposes and those purposes statedherein and therein. This application further claims priority under 35U.S.C. §120 as a continuation-in-part of the non-provisional patentapplication titled “Microfluidic Particle-Analysis Systems”, by Chou etal., filed on Mar. 31, 2003 (Atty. Docket No.: 139F.310US), which ishereby incorporated by reference for all purposes.

CROSS-REFERENCE TO PATENT APPLICATIONS

This application incorporates by reference in their entirety for allpurposes the following U.S. patent applications: Ser. No. 09/605,520,filed Jun. 27, 2000; Ser. No. 09/724,784, filed Nov. 28, 2000; Ser. No.09/724,967, filed Nov. 28, 2000; Ser. No. 09/796,378, filed Feb. 28,2001; Ser. No. 09/796,666, filed Feb. 28, 2001; Ser. No. 09/796,871,filed Feb. 28, 2001; Ser. No. 09/826,583, filed Apr. 6, 2001; and Ser.No. 09/724,784, filed Nov. 28, 2001, titled MICROFABRICATED ELASTOMERICVALVE AND PUMP SYSTEMS, and naming Marc A. Unger, Hou-Pu Chou, Todd A.Thorsen, Axel Scherer, Stephen R. Quake, Jian Liu, Mark L. Adams, andCarl L. Hansen as inventors.

CROSS-REFERENCES TO OTHER MATERIALS

This application incorporates by reference in their entirety for allpurposes the following publications: Joe Sambrook and David Russell,Molecular Cloning: A Laboratory Manual (3^(rd) ed. 2000); and R. IanFreshney, Culture of Animal Cells: A Manual of Basic Technique (4^(th)ed. 2000).

FIELD OF THE INVENTION

The invention relates to systems for the manipulation and/or detectionof particles. More particularly, the invention relates to microfluidicsystems for the manipulation and/or detection of particles, such ascells and/or beads.

BACKGROUND OF THE INVENTION

The ability to perform molecular and cellular analyses of biologicalsystems has grown explosively over the past three decades. Inparticular, the advent and refinement of molecular and cellulartechniques, such as DNA sequencing, gene cloning, monoclonal antibodyproduction, cell transfection, amplification techniques (such as PCR),and transgenic animal formation, have fueled this explosive growth.These techniques have spawned an overwhelming number of identifiedgenes, encoded proteins, engineered cell types, and assays for studyingthese genes, proteins, and cell types. As the number of possiblecombinations of samples, reagents, and assays becomes nearlyincalculable, it has become increasingly apparent that novel approachesare necessary even to begin to make sense of this complexity, especiallywithin reasonable temporal and monetary limitations.

One approach to these difficulties has been to reduce the scale ofassays. Accordingly, substantial effort has been directed to developingassay methods and instrumentation for high-density microtiter plates.However, very small assay volumes in high-density microtiter plates,particularly assays with cells, may suffer from a number ofshortcomings. For example, cells may be lost easily from wells, may beharmed by rapid fluid evaporation, may contaminate nearby wells, and maybe difficult to remove efficiently from wells for additional analysis orculture. Thus, there is a need for systems that can effectivelymanipulate and analyze cells and other small particles, such as beads,in small volumes.

SUMMARY OF THE INVENTION

The invention provides systems, including apparatus, methods, and kits,for the microfluidic manipulation and/or detection of particles, such ascells and/or beads.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing potential temporal relationships betweenmethod steps for manipulation and/or detection of particles in amicrofluidic system, in accordance with aspects of the invention.

FIG. 2A is a top plan view of a microfluidic system for retaining andanalyzing a subset of input particles, in accordance with aspects of theinvention.

FIG. 2B is a top plan view of another microfluidic system for retainingand analyzing a subset of input particles, in accordance with aspects ofthe invention.

FIG. 3 is a fragmentary, top plan view of yet another microfluidicsystem for retaining and analyzing a subset of input particles, inaccordance with aspects of the invention.

FIG. 4 is a view of the system of FIG. 3 during particle positioning andretention, illustrating the various flow paths followed by particles, inaccordance with aspects of the invention.

FIG. 5 is a fragmentary, top plan view of a microfluidic system forpositioning and retaining a group of particles, and for perfusing theretained group with selected reagents, in accordance with aspects of theinvention.

FIG. 6 is a photographic image of a portion of a chip fabricatedaccording to the system of FIG. 5, in accordance with aspects of theinvention.

FIG. 7 is a schematic rendition of the image of FIG. 6, illustratingpaths of fluid flow and particle movement relative to aparticle-retention or -capture chamber, in accordance with aspects ofthe invention.

FIG. 8 is a full top plan view of the system of FIG. 5.

FIG. 9 is a photographic image of cells in a retention chamber, afterexposure to Trypan blue to stain lysed cells, but before cell fixation,in accordance with aspects of the invention.

FIG. 10 is another photographic image of the cells and chamber of FIG.9, after exposure to methanol to lyse and fix the cells, in accordancewith aspects of the invention.

FIG. 11 is yet another photographic image of the cells and chamber ofFIG. 9, after exposure to 1) methanol to lyse and fix the cells, 2)Trypan blue to stain lysed cells, and 3) a wash buffer to remove excessTrypan blue, in accordance with aspects of the invention.

FIG. 11A is a fragmentary, top plan view of a microfluidic system formeasuring cell-cell communication, based on a duplicated version of thesystem of FIG. 8, in accordance with aspects of the invention.

FIG. 11B is a top plan view of selected portions of an alternativeembodiment of the system of FIG. 11A, in accordance with aspects of theinvention.

FIG. 11C is a top plan view of a two-dimensional array of particlecapture chambers that may be used in a microfluidic system, inaccordance with aspects of the invention.

FIG. 12 is a fragmentary, top plan view of a microfluidic system forretaining and perfusing two sets of particles in parallel, in accordancewith aspects of the invention.

FIG. 13 is a view of selected portions of the system of FIG. 12,illustrating paths for fluid flow and particle movement relative to twoadjacent retention chambers, in accordance with aspects of theinvention.

FIG. 13A is a top plan view of a microfluidic system for retaining twoparticles at spaced sites in a channel and perfusing the retainedparticles with distinct reagents, in accordance with aspects of theinvention.

FIG. 13B is a top plan view of selected portions of the system of FIG.13A, in accordance with aspects of the invention.

FIG. 13C is a top plan view of selected portions of an alternativeembodiment of the system of FIG. 13A, in accordance with aspects of theinvention.

FIG. 13D is a photograph of two beads being exposed to green dyedelivered by spaced treatment mechanisms, using a chip constructedaccording to the system of FIG. 13A, in accordance with aspects of theinvention.

FIG. 13E is another photograph of the two beads of FIG. 13D duringexposure to a red dye and a green dye delivered by spaced treatmentmechanisms, in accordance with aspects of the invention.

FIG. 13F is yet another photograph of the two beads of FIG. 13D duringexposure to a red dye and a yellow dye delivered by spaced treatmentmechanisms, in accordance with aspects of the invention.

FIG. 13G is a photograph of two cells held at separate retention sitesin a chip constructed according to the system of FIG. 13A, in accordancewith aspects of the invention.

FIG. 13H is a photograph of the two cells of FIG. 13G during exposure toa blue dye delivered by spaced treatment mechanisms, in accordance withaspects of the invention.

FIG. 13I is a photograph of the two cells of FIG. 13G during treatmentof only one of the cells with an organic fixative, in accordance withaspects of the invention.

FIG. 13J is a photograph of the two cells of FIG. 13I, after fixation ofthe one cell and during exposure to a blue dye, delivered by spacedtreatment mechanisms, in accordance with aspects of the invention.

FIG. 13K is a photograph of two fluorescent beads held at two retentionsites and individually exposed to a fluorescent and a chromophoric dyedelivered by spaced treatment mechanisms, but without the use of aspacer buffer, using a chip constructed according to the system of FIG.13A, in accordance with aspects of the invention.

FIG. 13L is a fragmentary, top plan view of a microfluidic system havingseparately addressable sets of linear trap arrays, in accordance withaspects of the invention.

FIG. 14 is a top plan view of a microfluidic system for retaining anarray of particles in series and for perfusing members of this arrayseparately and in parallel, in accordance with aspects of the invention.

FIG. 15 is a top plan view of selected portions of the system of FIG.14, illustrating fluid-layer and control-layer networks for treatingretained particles separately and in parallel, in accordance withaspects of the invention.

FIG. 16 is a top plan view of portions of a single retention networkfrom the system of FIG. 14, illustrating selected paths of fluid flow,in accordance with aspects of the invention.

FIG. 17 is a fragmentary, top plan view of a microfluidic device forforming an array of single particles or groups of particles, inaccordance with aspects of the invention.

FIG. 18 is a pair of fragmentary, top plan schematic views of amicrofluidic device for forming an array of retained particles that maybe transferred to an array of separate sites, illustrating particleretention and transfer configurations, on the left and rightrespectively, in accordance with aspects of the invention.

FIG. 19 is a pair of fragmentary, top plan schematic views of anothermicrofluidic device for forming an array of retained particles that maybe transferred to an array of separate sites, illustrating particleretention and transfer configurations, on the left and rightrespectively, in accordance with aspects of the invention.

FIG. 20 is fragmentary, top plan schematic view of yet anothermicrofluidic device for forming an array of retained particles that maybe transferred to an array of separate sites, in accordance with aspectsof the invention.

FIG. 21 is a composite of top plan and sectional views showing selectedportions of a microfluidic system for retaining particles using aparticle-retention chamber that is fully spaced from the floor of thesystem, in accordance with aspects of the invention.

FIG. 22 is a composite of top plan and sectional views, and aphotographic image, showing selected portions of a microfluidic systemfor retaining particles using a particle-retention chamber that ispartially spaced from the floor of the system, in accordance withaspects of the invention.

FIG. 23 is a composite of top plan and sectional views, and twophotographic images, showing selected portions of another microfluidicsystem for retaining particles using a particle-retention chamber thatis fully spaced from the floor of the system, in accordance with aspectsof the invention.

FIG. 24 is a fragmentary, top plan view of a reusable microfluidicsystem for repeated retention, treatment, and release of singleparticles, in accordance with aspects of the invention.

FIG. 25 is a view of selected portions of the system of FIG. 24,particularly a particle release mechanism, in accordance with aspects ofthe invention.

FIG. 26 is a fragmentary, top plan view of a reusable microfluidicsystem for repeated retention, treatment, and release of groups ofparticles, in accordance with aspects of the invention.

FIG. 27 is a view of selected portions of the systems of FIGS. 24 and26, particularly a particle collection mechanism, in accordance withaspects of the invention.

FIG. 28 is a fragmentary, top plan view of an input mechanism thatincludes a particle suspension mechanism, in accordance with aspects ofthe invention.

FIG. 29 is a fragmentary, top plan view of an adjustable dilutionmechanism, in accordance with aspects of the invention.

FIG. 30 is a fragmentary, top plan view of another adjustable dilutionmechanism, in accordance with aspects of the invention.

FIG. 31 is a top plan view of a microfluidic system having a sortingmechanism based on centrifugal force, in accordance with aspects of theinvention.

FIG. 32 is a fragmentary view of the system of FIG. 31, showing thesorting mechanism in greater detail, in accordance with aspects of theinvention.

FIG. 33 is a fragmentary, top plan view of another microfluidic systemhaving a sorting mechanism based on centrifugal force, in accordancewith aspects of the invention.

FIG. 34 is a top plan view of a yet another microfluidic system having asorting mechanism based on centrifugal force, in accordance with aspectsof the invention.

FIG. 35 is a fragmentary view of the system of FIG. 34, showing thesorting mechanism in greater detail.

FIG. 36 is a photographic image of fluorescent beads and particles beingseparated by the sorting mechanism of FIGS. 34 and 35.

FIG. 37 is a graph plotting the ratio of cells to beads over time duringsorting with the system of FIGS. 34 and 35.

FIG. 38 is a graph plotting the ratio of cells to beads over time duringsorting with the system of FIGS. 31 and 32.

FIGS. 39-43 are top plan composite views of various cell-chambernetworks for microfluidic manipulation of cells, in accordance withaspects of the invention.

FIG. 44 is a top plan view of a microfluidic system with a parallelarray of separate, isolatable cell-chamber networks, in accordance withaspects of the invention.

FIG. 45 is a top plan view of a microfluidic system with an isolatablecell chamber that may be fed or bypassed by a parallel fluidic circuit,in accordance with aspects of the invention.

FIG. 46 is a top plan view of a microfluidic system having a cellchamber that forms a loop, in accordance with aspects of the invention

FIG. 47 is a top plan view of a microfluidic system in which particleand reagent networks intersect at a common cell chamber, in accordancewith aspects of the invention.

FIGS. 48 and 49 are photographic images of filtering mechanisms withsize-selective channels that are included in the reagent networks ofchips fabricated according to the system of FIG. 47.

FIG. 50 is a composite of two photographic images showing cells culturedin a cell chamber of a chip fabricated according to the system of FIG.47.

FIG. 50A is a fragmentary, top plan view of a system for depositingcells in a cell chamber, based on a nonlinear, asymmetrical flow path,in accordance with aspects of the invention.

FIG. 50B is a fragmentary, top plan view of a modified version of thesystem of FIG. 50A, in which reagent(s) may be recirculated through thecell chamber, in accordance with aspects of the invention.

FIG. 50C is a top plan view of a cell chamber having two distinctcompartments connected by a set of radially arrayed, size-selectivechannels, in accordance with aspects of the invention.

FIG. 50D is a top plan view of a version of the cell chamber of FIG.50C, modified to interconnect the two compartments more fully, inaccordance with aspects of the invention.

FIG. 51 is an isometric schematic view of a microfluidic system forperforming electrophysiological analysis on an array of cells, inaccordance with aspects of the invention.

FIG. 52 is a top plan view of a microfluidic system for performingelectrophysiological analysis on a single cell, in accordance withaspects of the invention.

FIG. 53 is a fragmentary top plan view of a microfluidic system relatedto the system of FIG. 52, showing a modified focusing mechanism, inaccordance with aspects of the invention.

FIG. 54 is a top plan view of selected portions of the system of FIG. 52with a retained cell, in accordance with aspects of the invention.

FIG. 55 is a top plan view of selected portions of the system of FIG. 52during perfusion of a retained cell, in accordance with aspects of theinvention.

FIG. 56 is another top plan view of selected portions of the system ofFIG. 52, in accordance with aspects of the invention.

FIG. 57 is yet another top plan view of selected portions of the systemof FIG. 52, in accordance with aspects of the invention.

FIG. 58 is a photographic image of a portion of a chip fabricatedaccording to the system of FIG. 52.

FIG. 59 is an abstracted view of a microfluidic device for performingpatch-clamp analysis of cells, in accordance with aspects of theinvention.

FIG. 60 is a fragmentary top plan view of a microfluidic device forperforming patch-clamp analysis of multiple individual cells, inaccordance with aspects of the invention.

FIG. 61 is a graph showing 95% probability of successfully obtaining anelectrophysiological reading as a function of both the number ofapertures (channels) analyzed and the fraction of individual aperturesthat give a successful reading.

FIG. 62 is a fragmentary side elevation view of a microfluidic moldspin-coated with a first layer of patternable, selectively removablematerial, in accordance with aspects of the invention.

FIG. 63 is a fragmentary side elevation view of the mold of FIG. 62after patterned removal of the first layer, in accordance with aspectsof the invention.

FIG. 64 is a fragmentary side elevation view of the mold of FIG. 63spin-coated with a second layer of patternable, selectively removablematerial, in accordance with aspects of the invention.

FIG. 65 is a fragmentary side elevation view of the mold of FIG. 64after patterned removal of the second layer, in accordance with aspectsof the invention.

FIG. 66 is a fragmentary side elevation view of the mold of FIG. 65after heating at elevated temperatures to round remaining portions ofthe second layer, in accordance with aspects of the invention.

FIG. 67 is a fragmentary side elevation view of the mold of FIG. 66spin-coated with a third layer of patternable, selectively removablematerial, in accordance with aspects of the invention.

FIG. 68 is a fragmentary side elevation view of the mold of FIG. 67following patterned removal of the third layer, in accordance withaspects of the invention.

FIG. 69 is a fragmentary side elevation view of the mold of FIG. 68acting to mold complementary surface features of a fluid-layer membrane,in accordance with aspects of the invention.

FIG. 70 is a composite of photographic images of 1) a fluid-layer moldformed using the method depicted in FIGS. 62-68 and 2) a correspondingmolded chip formed from the fluid-layer mold, in accordance with aspectsof the invention.

FIG. 71 is a composite of photographic images of 1) a fluid-layer moldformed using the method depicted in FIGS. 62-68 and 2) a correspondingmolded chip formed partially from the fluid-layer mold, in accordancewith aspects of the invention.

FIG. 71A is a graph of fluorescence emission versus time for afluorophore being excited at different light intensities, in accordancewith aspects of the invention.

FIG. 71B is a schematic diagram of an embodiment of a method forincreasing the signal-to-noise ratio of a detected signal by modulationof an exciting light source and demodulation of the detected signal,based on the modulation, in accordance with aspects of the invention.

FIG. 71C is a pair of graphs of time-dependent measured noise andmeasured signal plus noise without (top) and with (bottom)implementation of the modulation-demodulation method of FIG. 71B in amicrofluidic system, in accordance with aspects of the invention.

FIG. 71D is a graph of measured fluorescence intensity versus time priorto and during cycles of exposure of a biotinylated bead to astreptavidin-dye conjugate in a microfluidic system, in accordance withaspects of the invention.

FIG. 71E is a graph of measured fluorescence intensity versus time priorto and during exposure of ionomcyin to a retained cell that waspreloaded with a calcium-sensor dye, using the method of FIG. 71B in amicrofluidic system, in accordance with aspects of the invention.

FIG. 71F is a graph of measured fluorescence intensity versus time at aposition in a microfluidic system prior to and during exposure to a dye,in accordance with aspects of the invention.

FIG. 72 is a time-lapse set of photographic images recordingsize-selective flow of blood cells through a microfluidic system, inaccordance with aspects of the invention.

FIG. 73 is diagram showing the structure of biotin and its mode ofbinding to streptavidin.

FIG. 74 is a time-lapse set of photographic images recording interactionof specific binding pairs on beads in a microfluidic system, inaccordance with aspects of the invention.

FIG. 75 is a time-lapse set of photographic images recording stimulationof ion flux in a microfluidic system, in accordance with aspects of theinvention.

FIG. 76 is a time-lapse set of photographic images recording apoptosisand necrosis in a microfluidic system, in accordance with aspects of theinvention.

FIGS. 77 and 78 are diagrams showing the structures andexcitation/emission spectra for membrane dyes used in the analysis ofExample 22.

FIG. 79 is a photographic image recording successful staining of acell's membrane in a non-microfluidic environment.

FIG. 80 is a time-lapse set of photographic images recording retentionof a single cell at a preselected site in a microfluidic system, inaccordance with aspects of the invention.

FIG. 81 is a time-lapse set of photographic images recording retentionof a group of cells at a preselected site in a microfluidic system, inaccordance with aspects of the invention.

FIG. 82 is a time-lapse set of photographic images recording entry of afluorescent cell into a retention chamber already holding several cells,in accordance with aspects of the invention.

FIG. 83 is a time-lapse set of photographic images recording fixationand staining of a retained cell in a microfluidic system, in accordancewith aspects of the invention.

FIG. 84 is a top plan view of a microfluidic system for analyzing asize-selected set of cells, in which the system includes seriallydisposed filtration and retention mechanisms, a perfusion mechanism, anda flow-based detection mechanism, in accordance with aspects of theinvention.

FIG. 85 is another top plan view of the microfluidic system of FIG. 84,showing identifying labels for reservoirs and valves, in accordance withaspects of the invention.

FIG. 86 is a top plan view of selected portions of the system of FIG.84, illustrating selected aspects including a filtration mechanism, inaccordance with aspects of the invention.

FIG. 87 is another top plan view of selected portions of the system ofFIG. 84, in accordance with aspects of the invention.

FIG. 88 is yet another top plan view of selected portions of the systemof FIG. 84, in accordance with aspects of the invention.

FIG. 89 is a top plan view of a perfusion device for exposing particlesto an array of different reagents or different reagent concentrations.

FIGS. 90 through 94 depict a top plan view of a device being used tomeasure chemotactic response of cells to a chemoattractant.

FIG. 95 is a close-up top plan view of a perfusion chamber withassociated valving system.

DETAILED DESCRIPTION

The invention provides systems, including apparatus, methods, and kits,for the microfluidic manipulation and/or analysis of particles, such ascells, viruses, organelles, beads, and/or vesicles. The invention alsoprovides microfluidic mechanisms for carrying out these manipulationsand analyses. These mechanisms may enable controlled input,movement/positioning, retention/localization, treatment, measurement,release, and/or output of particles. Furthermore, these mechanisms maybe combined in any suitable order and/or employed for any suitablenumber of times within a system. Accordingly, these combinations mayallow particles to be sorted, cultured, mixed, treated, and/or assayed,among others, as single particles, mixed groups of particles, arrays ofparticles, heterogeneous particle sets, and/or homogeneous particlesets, among others, in series and/or in parallel. In addition, thesecombinations may enable microfluidic systems to be reused. Furthermore,these combinations may allow the response of particles to treatment tobe measured on a shorter time scale than was previously possible.Therefore, systems of the invention may allow a broad range of cell andparticle assays, such as drug screens, cell characterizations, researchstudies, and/or clinical analyses, among others, to be scaled down tomicrofluidic size. Such scaled-down assays may use less sample andreagent, may be less labor intensive, and/or may be more informativethan comparable macrofluidic assays.

Further aspects of the invention are described in the followingsections: (I) microfluidic systems, (II) physical structures of fluidnetworks, (III) particles, (IV) input mechanisms, (V) positioningmechanisms, (VI) retention mechanisms, (VII) treatment mechanisms,(VIII) measurement mechanisms, (IX) release mechanisms, (X) outputmechanisms, (XI) cell culture mechanisms, (XII) particle-basedmanipulations, and (XIII) examples.

Microfluidic Systems

Definitions and Overview

Particle manipulations and analyses are performed in microfluidicsystems. A microfluidic system generally comprises any system in whichvery small volumes of fluid are stored and manipulated, generally lessthan about 500 μL, typically less than about 100 μL, and more typicallyless than about 10 μL. Microfluidic systems carry fluid in predefinedpaths through one or more microfluidic passages. A microfluidic passagemay have a minimum dimension, generally height or width, of less thanabout 200, 100, or 50 μm. Passages are described in more detail below inSection II.

Microfluidic systems may include one or more sets of passages thatinterconnect to form a generally closed microfluidic network. Such amicrofluidic network may include one, two, or more openings at networktermini, or intermediate to the network, that interface with theexternal world. Such openings may receive, store, and/or dispense fluid.Dispensing fluid may be directly into the microfluidic network or tosites external the microfluidic system. Such openings generally functionin input and/or output mechanisms, described in more detail in SectionsIV and X below, and may include reservoirs, described in more detail inSection II below.

Microfluidic systems also may include any other suitable features ormechanisms that contribute to fluid, reagent, and/or particlemanipulation or analysis. For example, microfluidic systems may includeregulatory or control mechanisms that determine aspects of fluid flowrate and/or path. Valves and/or pumps that may participate in suchregulatory mechanisms are described in more detail below in Section II.Alternatively, or in addition, microfluidic systems may includemechanisms that determine, regulate, and/or sense fluid temperature,fluid pressure, fluid flow rate, exposure to light, exposure to electricfields, magnetic field strength, and/or the like. Accordingly,microfluidic systems may include heaters, coolers, electrodes, lenses,gratings, light sources, pressure sensors, pressure transducers,microprocessors, microelectronics, and/or so on. Furthermore, eachmicrofluidic system may include one or more features that act as a codeto identify a given system. The features may include any detectableshape or symbol, or set of shapes or symbols, such as black-and-white orcolored barcode, a word, a number, and/or the like, that has adistinctive position, identity, and/or other property (such as opticalproperty).

Materials

Microfluidic systems may be formed of any suitable material orcombination of suitable materials. Suitable materials may includeelastomers, such as polydimethylsiloxane (PDMS); plastics, such aspolystyrene, polypropylene, polycarbonate, etc.; glass; ceramics;sol-gels; silicon and/or other metalloids; metals or metal oxides;biological polymers, mixtures, and/or particles, such as proteins(gelatin, polylysine, serum albumin, collagen, etc.), nucleic acids,microorganisms, etc.; and/or the like.

Exemplary materials for microfluidic systems are described in moredetail in the patent applications listed above under Cross-References,which are incorporated herein by reference.

Methods of Fabrication

Microfluidic systems, also referred to as chips, may have any suitablestructure. Such systems may be fabricated as a unitary structure from asingle component, or as a multi-component structure of two or morecomponents. The two or more components may have any suitable relativespatial relationship and may be attached to one another by any suitablebonding mechanism.

In some embodiments, two or more of the components may be fabricated asrelatively thin layers, which may be disposed face-to-face. Therelatively thin layers may have distinct thickness, based on function.For example, the thickness of some layers may be about 10 to 250 μm, 20to 200 μm, or about 50 to 150 μm, among others. Other layers may besubstantially thicker, in some cases providing mechanical strength tothe system. The thicknesses of such other layers may be about 0.25 to 2cm, 0.4 to 1.5 cm, or 0.5 to 1 cm, among others. One or more additionallayers may be a substantially planar layer that functions as a substratelayer, in some cases contributing a floor portion to some or allmicrofluidic passages.

Components of a microfluidic system may be fabricated by any suitablemechanism, based on the desired application for the system and onmaterials used in fabrication. For example, one or more components maybe molded, stamped, and/or embossed using a suitable mold. Such a moldmay be formed of any suitable material by micromachining, etching, softlithography, material deposition, cutting, and/or punching, amongothers. Alternatively, or in addition, components of a microfluidicsystem may be fabricated without a mold by etching, micromachining,cutting, punching, and/or material deposition.

Microfluidic components may be fabricated separately, joined, andfurther modified as appropriate. For example, when fabricated asdistinct layers, microfluidic components may be bonded, generallyface-to-face. These separate components may be surface-treated, forexample, with reactive chemicals to modify surface chemistry, withparticle binding agents, with reagents to facilitate analysis, and/or soon. Such surface-treatment may be localized to discrete portions of thesurface or may be relatively nonlocalized. In some embodiments, separatelayers may be fabricated and then punched and/or cut to produceadditional structure. Such punching and/or cutting may be performedbefore and/or after distinct components have been joined.

Exemplary methods for fabricating microfluidic systems are described inmore detail in the patent applications identified above underCross-References, which are incorporated herein by reference.

Physical Structures of Fluid Networks

Overview

Microfluidic systems may include any suitable structure(s) for theintegrated manipulation of small volumes of fluid, including movingand/or storing fluid, and particles associated therewith, for use inparticle assays. The structures may include passages, reservoirs, and/orregulators, among others.

Passages

Passages generally comprise any suitable path, channel, or duct through,over, or along which materials (e.g., fluid, particles, and/or reagents)may pass in a microfluidic system. Collectively, a set of fluidicallycommunicating passages, generally in the form of channels, may bereferred to as a microfluidic network. In some cases, passages may bedescribed as having surfaces that form a floor, a roof, and walls.Passages may have any suitable dimensions and geometry, including width,height, length, and/or cross-sectional profile, among others, and mayfollow any suitable path, including linear, circular, and/orcurvilinear, among others. Passages also may have any suitable surfacecontours, including recesses, protrusions, and/or apertures, and mayhave any suitable surface chemistry or permeability at any appropriateposition within a channel. Suitable surface chemistry may includesurface modification, by addition and/or treatment with a chemicaland/or reagent, before, during, and/or after passage formation.

In some cases, passages, and particularly channels, may be describedaccording to function. For example, passages may be described accordingto direction of material flow in a particular application, relationshipto a particular reference structure, and/or type of material carried.Accordingly, passages may be inlet passages (or channels), whichgenerally carry materials to a site, and outlet passages (or channels),which generally carry materials from a site. In addition, passages maybe referred to as particle passages (or channels), reagent passages (orchannels), focusing passages (or channels), perfusion passages (orchannels), waste passages (or channels), and/or the like.

Passages may branch, join, and/or dead-end to form any suitablemicrofluidic network. Accordingly, passages may function in particlepositioning, sorting, retention, treatment, detection, propagation,storage, mixing, and/or release, among others.

Further aspects of passages are included throughout this DetailedDescription, and in the patent applications identified above underCross-References, which are incorporated herein by reference.

Reservoirs

Reservoirs generally comprise any suitable receptacle or chamber forstoring materials (e.g., fluid, particles and/or reagents), before,during, between, and/or after processing operations (e.g., measurementand/or treatment). Reservoirs, also referred to as wells, may includeinput, intermediate, and/or output reservoirs. Input reservoirs maystore materials (e.g., fluid, particles, and/or reagents) prior toinputting the materials to a microfluidic network(s) portion of a chip.By contrast, intermediate reservoirs may store materials during and/orbetween processing operations. Finally, output reservoirs may storematerials prior to outputting from the chip, for example, to an externalprocessor or waste, or prior to disposal of the chip.

Further aspects of reservoirs are included in the patent applicationsidentified above under Cross-References, which are incorporated hereinby reference.

Regulators

Regulators generally comprise any suitable mechanism for generatingand/or regulating movement of materials (e.g., fluid, particles, and/orreagents). Suitable regulators may include valves, pumps, and/orelectrodes, among others. Regulators may operate by actively promotingflow and/or by restricting active or passive flow. Suitable functionsmediated by regulators may include mixing, sorting, connection (orisolation) of fluidic networks, and/or the like.

Further aspects of regulators, particularly the structure, fabrication,and operation of valves and pumps, are included in the patentapplications identified above under Cross-References, which areincorporated herein by reference, and in Section XIII, particularlyExample 8.

Particles

Overview

Microfluidic systems may be used to manipulate and/or analyze particles.A particle generally comprises any object that is small enough to beinputted and manipulated within a microfluidic network in associationwith fluid, but that is large enough to be distinguishable from thefluid. Particles, as used here, typically are microscopic ornear-microscopic, and may have diameters of about 0.005 to 100 μm, 0.1to 50 μm, or about 0.5 to 30 μm. Alternatively, or in addition,particles may have masses of about 10⁻²⁰ to 10⁻⁵ grams, 10⁻¹⁶ to 10⁻⁷grams, or 10⁻¹⁴ to 10⁻⁸ grams. Exemplary particles may include cells,viruses, organelles, beads, and/or vesicles, and aggregates thereof,such as dimers, trimers, etc.

Cells

Overview

Cells, as used here, generally comprise any self-replicating,membrane-bounded biological entity, or any nonreplicating,membrane-bounded descendant thereof. Nonreplicating descendants may besenescent cells, terminally differentiated cells, cell chimeras,serum-starved cells, infected cells, nonreplicating mutants, anucleatecells, etc.

Cells used as particles in microfluidic systems may have any suitableorigin, genetic background, state of health, state of fixation, membranepermeability, pretreatment, and/or population purity, among others.Origin of cells may be eukaryotic, prokaryotic, archae, etc., and may befrom animals, plants, fungi, protists, bacteria, and/or the like. Cellsmay be wild-type; natural, chemical, or viral mutants; engineeredmutants (such as transgenics); and/or the like. In addition, cells maybe growing, quiescent, senescent, transformed, and/or immortalized,among others, and cells may be fixed and/or unfixed. Living or dead,fixed or unfixed cells may have intact membranes, and/orpermeabilized/disrupted membranes to allow uptake of ions, labels, dyes,ligands, etc., or to allow release of cell contents. Cells may have beenpretreated before introduction into a microfluidic system by anysuitable processing steps. Such processing steps may include modulatortreatment, transfection (including infection, injection, particlebombardment, lipofection, coprecipitate transfection, etc.), processingwith assay reagents, such as dyes or labels, and/or so on. Furthermore,cells may be a monoculture, generally derived as a clonal populationfrom a single cell or a small set of very similar cells; may bepresorted by any suitable mechanism such as affinity binding, FACS, drugselection, etc.; and/or may be a mixed or heterogeneous population ofdistinct cell types.

Eukaryotic Cells

Eukaryotic cells, that is, cells having one or more nuclei, or anucleatederivatives thereof, may be obtained from any suitable source, includingprimary cells, established cells, and/or patient samples. Such cells maybe from any cell type or mixture of cell types, from any developmentalstage, and/or from any genetic background. Furthermore, eukaryotic cellsmay be adherent and/or nonadherent. Such cells may be from any suitableeukaryotic organism including animals, plants, fungi, and/or protists.

Eukaryotics cells may be from animals, that is, vertebrates orinvertebrates. Vertebrates may include mammals, that is, primates (suchas humans, apes, monkeys, etc.) or nonprimates (such as cows, horses,sheep, pigs, dogs, cats, marsupials, rodents, and/or the like).Nonmammalian vertebrates may include birds, reptiles, fish, (such astrout, salmon, goldfish, zebrafish, etc.), and/or amphibians (such asfrogs of the species Xenopus, Rana, etc.). Invertebrates may includearthropods (such as arachnids, insects (e.g., Drosophila), etc.),mollusks (such as clams, snails, etc.), annelids (such as earthworms,etc.), echinoderms (such as various starfish, among others),coelenterates (such as jellyfish, coral, etc.), porifera (sponges),platyhelminths (tapeworms), nemathelminths (flatworms), etc.

Eukaryotic cells may be from any suitable plant, such as monocotyledons,dicotyledons, gymnosperms, angiosperms, ferns, mosses, lichens, and/oralgae, among others. Exemplary plants may include plant crops (such asrice, corn, wheat, rye, barley, potatoes, etc.), plants used in research(e.g., Arabadopsis, loblolly pine, etc.), plants of horticultural values(ornamental palms, roses, etc.), and/or the like.

Eukaryotic cells may be from any suitable fungi, including members ofthe phyla Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota,Deuteromycetes, and/or yeasts. Exemplary fungi may include Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoralis, Neurosporacrassa, mushrooms, puffballs, imperfect fungi, molds, and/or the like.

Eukaryotic cells may be from any suitable protists (protozoans),including amoebae, ciliates, flagellates, coccidia, microsporidia,and/or the like. Exemplary protists may include Giardia lamblia,Entamoeba. histolytica, Cryptosporidium, and/or N. fowleri, amongothers.

Particles may include eukaryotic cells that are primary, that is, takendirectly from an organism or nature, without subsequent extended culturein vitro. For example, the cells may be obtained from a patient sample,such as whole blood, packed cells, white blood cells, urine, sputum,feces, mucus, spinal fluid, tumors, diseased tissue, bone marrow, lymph,semen, pleural fluid, a prenatal sample, an aspirate, a biopsy,disaggregated tissue, epidermal cells, keratinocytes, endothelial cells,smooth muscle cells, skeletal muscle cells, neural cells, renal cells,prostate cells, liver cells, stem cells, osteoblasts, and/or the like.Similar samples may be manipulated and analyzed from human volunteers,selected members of the human population, forensic samples, animals,plants, and/or natural sources (water, soil, air, etc.), among others.

Alternatively, or in addition, particles may include establishedeukaryotic cells. Such cells may be immortalized and/or transformed byany suitable treatment, including viral infection, nucleic acidtransfection, chemical treatment, extended passage and selection,radiation exposure, and/or the like. Such established cells may includevarious lineages such as neuroblasts, neurons, fibroblasts, myoblasts,myotubes, chondroblasts, chondrocytes, osteoblasts, osteocytes,cardiocytes, smooth muscle cells, epithelial cells, keratinocytes,kidney cells, liver cells, lymphocytes, granulocytes, and/ormacrophages, among others. Exemplary established cell lines may includeRat-1, NIH 3T3, HEK 293, COS1, COS7, CV-1, C2C12, MDCK, PC12, SAOS,HeLa, Schneider cells, Junkat cells, SL2, and/or the like.

Prokaryotic Cells

Particles may be prokaryotic cells, that is, self-replicating,membrane-bounded microorganisms that lack membrane-bound organelles, ornonreplicating descendants thereof. Prokaryotic cells may be from anyphyla, including Aquificae, Bacteroids, Chlorobia, Chrysogenetes,Cyanobacteria, Fibrobacter, Firmicutes, Flavobacteria, Fusobacteria,Proteobacteria, Sphingobacteria, Spirochaetes, Thermomicrobia, and/orXenobacteria, among others. Such bacteria may be gram-negative,gram-positive, harmful, beneficial, and/or pathogenic. Exemplaryprokaryotic cells may include E. coli, S. typhimurium, B subtilis, S.aureus, C. perfringens, V. parahaemolyticus, and/or B. anthracis, amongothers.

Viruses

Viruses may be manipulated and/or analyzed as particles in microfluidicsystems. Viruses generally comprise any microscopic/submicroscopicparasites of cells (animals, plants, fungi, protists, and/or bacteria)that include a protein and/or membrane coat and that are unable toreplicate without a host cell. Viruses may include DNA viruses, RNAviruses, retroviruses, virions, viroids, prions, etc. Exemplary virusesmay include HIV, RSV, rabies, hepatitis virus, Epstein-Barr virus,rhinoviruses, bacteriophages, prions that cause various diseases (CJD(Creutzfeld-Jacob disease, kuru, GSS (Gerstmann-Straussler-Scheinkersyndrome), FFI (Fatal Familial Insomnia), Alpers syndrome, etc.), and/orthe like.

Organelles

Organelles may be manipulated and/or analyzed in microfluidic systems.Organelles generally comprise any particulate component of a cell. Forexample, organelles may include nuclei, Golgi apparatus, lysosomes,endosomes, mitochondria, peroxisomes, endoplasmic reticulum, phagosomes,vacuoles, chloroplasts, etc.

Beads

Particle assays may be performed with beads. Beads generally compriseany suitable manufactured particles. Beads may be manufactured frominorganic materials, or materials that are synthesized chemically,enzymatically and/or biologically. Furthermore, beads may have anysuitable porosity and may be formed as a solid or as a gel. Suitablebead compositions may include plastics (e.g., polystyrene), dextrans,glass, ceramics, sol-gels, elastomers, silicon, metals, and/orbiopolymers (proteins, nucleic acids, etc.). Beads may have any suitableparticle diameter or range of diameters. Accordingly, beads may be asubstantially uniform population with a narrow range of diameters, orbeads may be a heterogeneous population with a broad range of diameters,or two or more distinct diameters.

Beads may be associated with any suitable materials. The materials mayinclude compounds, polymers, complexes, mixtures, phages, viruses,and/or cells, among others. For example, the beads may be associatedwith a member of a specific binding pair (see Section VI), such as areceptor, a ligand, a nucleic acid, a member of a compound library,and/or so on. Beads may be a mixture of distinct beads, in some casescarrying distinct materials. The distinct beads may differ in anysuitable aspect(s), such as size, shape, an associated code, and/ormaterial carried by the beads. In some embodiments, the aspect mayidentify the associated material. Codes are described further in SectionXII below.

Vesicles

Particles may be vesicles. Vesicles generally comprise any noncellularlyderived particle that is defined by a lipid envelope. Vesicles mayinclude any suitable components in their envelope or interior portions.Suitable components may include compounds, polymers, complexes,mixtures, aggregates, and/or particles, among others. Exemplarycomponents may include proteins, peptides, small compounds, drugcandidates, receptors, nucleic acids, ligands, and/or the like.

Input Mechanisms

Overview

Microfluidic systems may include one or more input mechanisms thatinterface with the microfluidic network(s). An input mechanism generallycomprises any suitable mechanism for inputting material(s) (e.g.,particles, fluid, and/or reagents) to a microfluidic network of amicrofluidic chip, including selective (that is, component-by-component)and/or bulk mechanisms.

Internal/External Sources

The input mechanism may receive material from internal sources, that is,reservoirs that are included in a microfluidic chip, and/or externalsources, that is, reservoirs that are separate from, or external to, thechip.

Input mechanisms that input materials from internal sources may use anysuitable receptacle to store and dispense the materials. Suitablereceptacles may include a void formed in the chip. Such voids may bedirectly accessible from outside the chip, for example, through a holeextending from fluidic communication with a fluid network to an externalsurface of the chip, such as the top surface. The receptacles may have afluid capacity that is relatively large compared to the fluid capacityof the fluid network, so that they are not quickly exhausted. Forexample, the fluid capacity may be at least about 1, 5, 10, 25, 50, or100 μL. Accordingly, materials may be dispensed into the receptaclesusing standard laboratory equipment, if desired, such as micropipettes,syringes, and the like.

Input mechanisms that input materials from external sources also may useany suitable receptacle and mechanism to store and dispense thematerials. However, if the external sources input materials directlyinto the fluid network, the external sources may need to interfaceeffectively with the fluid network, for example, using contact and/ornoncontact dispensing mechanisms. Accordingly, input mechanisms fromexternal sources may use capillaries or needles to direct fluidprecisely into the fluid network. Alternatively, or in addition, inputmechanisms from external sources may use a noncontact dispensingmechanism, such as “spitting,” which may be comparable to the action ofan inkjet printer. Furthermore, input mechanisms from external sourcesmay use ballistic propulsion of particles, for example, as mediated by agene gun.

Facilitating Mechanisms

The inputting of materials into the microfluidics system may befacilitated and/or regulated using any suitable facilitating mechanism.Such facilitating mechanisms may include gravity flow, for example, whenan input reservoir has greater height of fluid than an output reservoir.Facilitating mechanisms also may include positive pressure to pushmaterials into the fluidic network, such as mechanical or gas pressure,or centrifugal force; negative pressure at an output mechanism to drawfluid toward the output mechanism; and/or a positioning mechanism actingwithin the fluid network. The positioning mechanism may include a pumpand/or an electrokinetic mechanism. Positioning mechanisms are furtherdescribed below, in Section V. In some embodiments, the facilitatingmechanism may include a suspension mechanism to maintain particles suchas cells in suspension prior to inputting, for example, as described inExample 7.

Positioning Mechanisms

Overview

Microfluidic systems may include one or more positioning mechanisms. Apositioning mechanism generally comprises any mechanism for placingparticles at preselected positions on the chip after inputting, forexample, for retention, growth, treatment, and/or measurement, amongothers. Positioning mechanisms may be categorized without limitation invarious ways, for example, to reflect their origins and/or operationalprinciples, including direct and/or indirect, fluid-mediated and/ornon-fluid-mediated, external and/or internal, and so on. Thesecategories are not mutually exclusive. Thus, a given positioningmechanism may position a particle in two or more ways; for example,electric fields may position a particle directly (e.g., viaelectrophoresis) and indirectly (e.g., via electroosmosis).

The positioning mechanisms may act to define particle positionlongitudinally and/or transversely. The term “longitudinal position”denotes position parallel to or along the long axis of a microfluidicchannel and/or a fluid flow stream within the channel. In contrast, theterm “transverse position” denotes position orthogonal to the long axisof a channel and/or an associated main fluid flow stream. Bothlongitudinal and transverse positions may be defined locally, byequating “long axis” with “tangent” in curved channels.

The positioning mechanisms may be used alone and/or in combination. Ifused in combination, the mechanisms may be used serially (i.e.,sequentially) and/or in parallel (i.e., simultaneously). For example, anindirect mechanism such as fluid flow may be used for rough positioning,and a direct mechanism such as optical tweezers may be used for finalpositioning (and/or subsequent retention, as described elsewhere).

The remainder of this section describes without limitation a variety ofexemplary positioning mechanisms, sorted roughly as direct and indirectmechanisms.

Direct Positioning Mechanisms

Direct positioning mechanisms generally comprise any mechanisms in whicha force acts directly on a particle(s) to position the particle(s)within a microfluidic network. Direct positioning mechanisms may bebased on any suitable mechanism, including optical, electrical,magnetic, and/or gravity-based forces, among others. Optical positioningmechanisms use light to mediate or at least facilitate positioning ofparticles. Suitable optical positioning mechanisms include “opticaltweezers,” which use an appropriately focused and movable light sourceto impart a positioning force on particles. Electrical positioningmechanisms use electricity to position particles. Suitable electricalmechanisms include “electrokinesis,” that is, the application of voltageand/or current across some or all of a microfluidic network, which may,as mentioned above, move charged particles directly (e.g., viaelectrophoresis) and/or indirectly, through movement of ions in fluid(e.g., via electroosmosis). Magnetic positioning mechanisms usemagnetism to position particles based on magnetic interactions. Suitablemagnetic mechanisms involve applying a magnetic field in or around afluid network, to position particles via their association withferromagnetic and/or paramagnetic materials in, on, or about theparticles. Gravity-based positioning mechanisms use the force of gravityto position particles, for example, to contact adherent cells with asubstrate at positions of cell culture.

Indirect Positioning Mechanisms

Indirect positioning mechanisms generally comprise any mechanisms inwhich a force acts indirectly on a particle(s), for example, via fluid,to move the particle(s) within a microfluidic network, longitudinallyand/or transversely.

Longitudinal Indirect Positioning Mechanisms

Longitudinal indirect positioning mechanisms generally may be createdand/or regulated by fluid flow along channels and/or other passages.Accordingly, longitudinal positioning mechanisms may be facilitatedand/or regulated by valves and/or pumps that regulate flow rate and/orpath. In some cases, longitudinal positioning mechanisms may befacilitated and/or regulated by electroosmotic positioning mechanisms.Alternatively, or in addition, longitudinal positioning mechanisms maybe input-based, that is, facilitated and/or regulated by inputmechanisms, such as pressure or gravity-based mechanisms, including apressure head created by unequal heights of fluid columns.

Transverse Indirect Positioning Mechanisms

Transverse indirect positioning mechanisms generally may be createdand/or regulated by fluid flow streams at channel junctions, laterallydisposed regions of reduced fluid flow, and/or channel bends. Channeljunctions may be unifying sites or dividing sites, based on the numberof channels that carry fluid to the sites relative to the number thatcarry fluid away from the sites. Transverse indirect positioningmechanisms may be based on laminar flow, stochastic partitioning, and/orcentrifugal force, among others.

Laminar Flow-Based Transverse Positioning Mechanisms

Transverse positioning of particles and/or reagents in a microfluidicsystem may be mediated at least in part by a laminar flow-basedmechanism. Laminar flow-based mechanisms generally comprise anypositioning mechanism in which the position of an input flow streamwithin a channel is determined by the presence, absence, and/or relativeposition(s) of additional flow streams within the channel. Such laminarflow-based mechanisms may be defined by a channel junction(s) that is aunifying site, at which inlet flow streams from two, three, or morechannels, flowing toward the junction, unify to form a smaller number ofoutlet flow streams, preferably one, flowing away from the junction. Dueto the laminar flow properties of flowstreams on a microfluidic scale,the unifying site may maintain the relative distribution of inlet flowstreams after they unify as laminar outlet flow streams. Accordingly,particles and/or reagents may remain localized to any selected one ormore of the laminar flow streams, based on which inlet channels carryparticles and/or reagents, thus positioning the particles and/orreagents transversely.

The relative size (or flow rate) and position of each inlet flow streammay determine both transverse position and relative width of flowstreams that carry particles and/or reagents. For example, an inlet flowstream for particles/reagents that is relatively small (narrow), flankedby two larger (wider) flow streams, may occupy a narrow central positionin a single outlet channel. By contrast, an inlet flow stream forparticles/reagents that is relatively large (wide), flanked by acomparably sized flow stream and a smaller (narrower) flow stream, mayoccupy a wider position that is biased transversely toward the smallerflow stream. In either case, the laminar flow-based mechanism may becalled a focusing mechanism, because the particles/reagents are“focused” to a subset of the cross-sectional area of outlet channels.Laminar flow-based mechanisms may be used to individually addressparticles and/or reagents to plural distinct retention sites. Exemplarylaminar flow-based positioning mechanisms are further described below,in Examples 2-4, 7, 9, 11, and 26, among others.

A laminar flow-based mechanism may be a variable mechanism to vary thetransverse position of particles/reagents. As described above, therelative contribution of each inlet flow stream may determine thetransverse position of particles/reagents flow streams. Altered flow ofany inlet flow stream may vary its contribution to the outlet flowstream(s), shifting particles/reagents flow streams accordingly. In anextreme case, referred to as a perfusion mechanism, a reagent (orparticle) flow stream may be moved transversely, either in contact with,or spaced from, retained particles (reagents), based on presence orabsence of flow from an adjacent inlet flow stream. Such a mechanismalso may be used to effect variable or regulated transverse positioningof particles, for example, to direct particles to retention sites havingdifferent transverse positions. Exemplary variable or regulatedtransverse positioning mechanisms, referred to as perfusion mechanisms,are further described below, in Examples 2-4, 6, 7, 11, and 26, amongothers.

Stochastic Transverse Positioning Mechanisms

Transverse positioning of particles and/or reagents in a microfluidicsystem may be mediated at least in part by a stochastic (or portionedflow) positioning mechanism. Stochastic transverse positioningmechanisms generally comprise any positioning mechanism in which an atleast partially randomly selected subset of inputted particles orreagent is distributed laterally away from a main flow stream to aregion of reduced fluid flow within a channel (or, potentially, to adistinct channel). The region of reduced flow may promote particleretention, treatment, detection, minimize particle damage, and/orpromote particle contact with a substrate. Stochastic positioningmechanisms may be determined by dividing flow sites and/or locallywidened channels, among others.

Dividing flow sites may effect stochastic positioning by forming regionsof reduced fluid flow rate. Dividing flow sites generally include anychannel junction at which inlet flow streams from one (preferably) ormore inlet channels are divided into a greater number of outletchannels, including two, three, or more, channels. Such dividing sitesmay deliver a subset of particles, which may be selected stochasticallyand/or based on a property of the particles (such as mass), to a regionof reduced flow rate or quasi-stagnant flow formed at or near thejunction. The fraction of particles represented by the subset may bedependent upon the relative flow directions of the outlet channelsrelative to the inlet channels. These flow directions may be generallyorthogonal to an inlet flow stream, being directed in oppositedirections, to form a “T-junction.” Alternatively, outlet flowdirections may form angles of less than and/or greater than 90°.Exemplary reduced-velocity, dividing-flow positioning mechanisms arefurther described below, in Examples 1, 2, 3, 4, 6, 7, and 26, amongothers.

The dividing-flow positioning mechanism, with two or more outletchannels, may be used as a portioned-flow mechanism. Specifically,fluid, particles, and/or reagents carried to the channel junction may beportioned according to fluid flow through the two or more outletchannels. Accordingly, the fractional number or volume of particles orreagent that enters the two or more channels may be regulated by therelative sizes of the channels and/or the flow rate of fluid through thechannels, which in turn may be regulated by valves, or other suitableflow regulatory-mechanisms. In a first set of embodiments, outletchannels may be of very unequal sizes, so that only a small fraction ofparticle and/or reagents are directed to the smaller channel. In asecond set of embodiments, valves may be used to forms desired dilutionsof reagents. In a third set of embodiments, valves may be used toselectively direct particles to one of two or more fluid paths. Examplesof these three sets of embodiments are further described below, inExamples 11, 8, and 7, respectively.

Locally widened channels may promote stochastic positioning by producingregions of decreased flow rate lateral to a main flow stream. Thedecreased flow rate may deposit a subset of inputted particles at aregion of decreased flow rate. Such widened channels may includenonlinear channels that curve or bend at an angle. Alternatively, or inaddition, widened regions may be formed by recesses formed in a channelwall(s), chambers that intersect channels, and/or the like, particularlyat the outer edge of a curved or bent channel. Exemplary locally widenedchannels that promote stochastic transverse positioning are describedfurther in Example 10.

Centrifugal-Force-Based Transverse Positioning Mechanisms

Transverse positioning of particles and/or reagents also may be mediatedat least in part by a centrifugal positioning mechanism. In centrifugalpositioning mechanisms, particles may experience a centrifugal forcedetermined by a change in velocity, for example, by moving through abend in a fluid path. Size and/or density of particles may determine therate of velocity change, distributing distinct sizes and/or densities ofparticle to distinct transverse positions. Exemplary centrifugalpositioning mechanisms are further described below, in Example 9.

Retention Mechanisms

Overview

Microfluidic systems may include one or more retention mechanisms. Aretention mechanism generally comprises any suitable mechanism forretaining (or holding, capturing, or trapping) particles at preselectedpositions or regions of microfluidic networks, including single orplural mechanisms, operating in series and/or in parallel. Retentionmechanisms may act to overcome the positioning force exerted by fluidflow. Furthermore, retention mechanisms, also referred to as capture ortrapping mechanisms, may retain any suitable number of particles,including single particles or groups/populations of particles. Suitableretention mechanisms may be based on physical barriers coupled withflow, chemical interactions, vacuum forces, fluid flow in a loop,gravity, centrifugal forces, magnetic forces, electrical forces, and/oroptically generated forces, among others.

Retention mechanisms may be selective or nonselective. Selectivemechanisms may be fractionally selective, that is, retaining less thanall (a subset of) inputted particles. Fractionally selective mechanismsmay rely at least in part on stochastic positioning mechanisms, such asthat exemplified in Example 2. Alternatively, or in addition, selectivemechanisms may be particle-dependent, that is, retaining particles basedon one or more properties of the inputted particle, such as size,surface chemistry, density, magnetic character, electrical charge,optical property (such as refractive index), and/or the like.

Physical Barrier-Based Retention Mechanisms

Retention mechanisms may be based at least partially on particle contactwith any suitable physical barrier(s) disposed in a microfluidicnetwork. Such particle-barrier contact generally restricts longitudinalparticle movement along the direction of fluid flow, producingflow-assisted retention. Flow-assisted particle-barrier contact also mayrestrict side-to-side/orthogonal (transverse) movement. Suitablephysical barriers may be formed by protrusions that extend inward fromany portion of a channel or other passage (that is, walls, roof, and/orfloor). For example, the protrusions may be fixed and/or movable,including columns, posts, blocks, bumps, walls, and/orpartially/completely closed valves, among others. Some physicalbarriers, such as valves, may be movable or regulatable. Alternatively,or in addition, a physical barrier may be defined by a recess(es) formedin a channel or other passage, or by a fluid-permeable membrane. Otherphysical barriers may be formed based on the cross-sectional dimensionsof passages. For example, size-selective channels may retain particlesthat are too large to enter the channels. (Size-selective channels alsomay be referred to as filter channels, microchannels, orparticle-restrictive or particle-selective channels.)

Further aspects of physical barriers and size-selective channels aredescribed below in Section XIII, and in the patent applications listedin the Cross-References, which are incorporated herein by reference.

Chemical Retention Mechanisms

Chemical retention mechanisms may retain particles based on chemicalinteractions. The chemical interactions may be covalent and/ornoncovalent interactions, including ionic, electrostatic, hydrophobic,van der Waals, and/or metal coordination interactions, among others.Chemical interactions may retain particles selectively and/ornonselectively. Selective and nonselective retention may be based onspecific and/or nonspecific chemical interactions between particles andpassage surfaces.

Chemical interactions may be specific. Specific mechanisms may usespecific binding pairs (SBPs), for example, with first and second SBPmembers disposed on particles and passage surfaces, respectively.Exemplary SBPs may include biotin/avidin, antibody/antigen,lectin/carbohydrate, etc. These and additional exemplary SBPs are listedbelow in Table 1, with the designations of first and second beingarbitrary. SBP members may be disposed locally within microfluidicnetworks before, during and/or after formation of the networks. Forexample, surfaces of a substrate and/or a fluid layer component may belocally modified by adhesion/attachment of a SBP member before thesubstrate and fluid layer component are joined. Alternatively, or inaddition, an SBP member may be locally associated with a portion of amicrofluidic network after the network has been formed, for example, bylocal chemical reaction of the SBP member with the network (such ascatalyzed by local illumination with light).

TABLE 1 Representative Specific Binding Pairs First SBP Member SecondSBP Member Antigen antibody Biotin avidin or streptavidin Carbohydratelectin or carbohydrate receptor DNA antisense DNA or DNA-binding proteinenzyme substrate or inhibitor enzyme Histidine NTA (nitrilotriaceticacid) IgG protein A or protein G RNA antisense RNA

Chemical interactions also may be relatively nonspecific. Nonspecificinteraction mechanisms may rely on local differences in the surfacechemistry of microfluidic networks. Such local differences may becreated before, during and/or after passage/microfluidic networkformation, as described above. The local differences may result fromlocalized chemical reactions, for example, to create hydrophobic orhydrophilic regions, and/or localized binding of materials. The boundmaterials may include poly-L-lysine, poly-D-lysine, polyethylenimine,albumin, gelatin, collagen, laminin, fibronectin, entactin, vitronectin,fibrillin, elastin, heparin, keratan sulfate, heparan sulfate,chondroitin sulfate, hyaluronic acid, and/or extracellular matrixextracts/mixtures, among others.

Other Retention Mechanisms

Other retention mechanisms may be used alternatively, or in addition to,physical barrier-based and/or chemical interaction-based retention. Someor all of these mechanisms, and/or the mechanisms described above, mayrely at least partially on friction between particles and passages toassist retention.

Retention mechanisms may be based on vacuum forces, fluid flow, and/orgravity. Vacuum-based retention mechanisms may exert forces that pullparticles into tighter contact with passage surfaces, for example, usinga force directed outwardly from a channel. Application of a vacuum,and/or particle retention, may be assisted by an aperture/orifice in thewall of a channel or other passage. By contrast, fluid flow-basedretention mechanisms may produce fluid flow paths, such as loops, thatretain particles. These fluid flow paths may be formed by a closedchannel-circuit having no outlet (e.g., by valve closure and activepumping), and/or by an eddy, such as that produced by generally circularfluid-flow within a recess. Gravity-based retention mechanisms may holdparticles against the bottom surfaces of passages, thus combining withfriction to restrict particle movement. Gravity-based retention may befacilitated by recesses and/or reduced fluid flow rates. Further aspectsof vacuum-based and fluid flow-based retention mechanisms are describedbelow in Examples 11 and 12, and Example 10, respectively.

Retention mechanisms may be based on centrifugal forces, magneticforces, and/or optically generated forces. Retention mechanisms based oncentrifugal force may retain particles by pushing the particle againstpassage surfaces, typically by exerting a force on the particles that isgenerally orthogonal to fluid flow. Such forces may be exerted bycentrifugation of a microfluidic chip and/or by particle movement withina fluid flow path (see Example 9). Magnetic force-based retentionmechanisms may retain particles using magnetic fields, generatedexternal and/or internal to a microfluidic system. The magnetic fieldmay interact with ferromagnetic and/or paramagnetic portions ofparticles. For example, beads may be formed at least partially offerromagnetic materials, or cells may include surface-bound orinternalized ferromagnetic particles. Electrical force-based retentionmechanisms may retain charged particles and/or populations usingelectrical fields. By contrast, retention mechanisms that operate basedon optically generated forces may use light to retain particles. Suchmechanisms may operate based on the principal of optical tweezers, amongothers.

Another form of retention mechanism is a blind-fill channel, where achannel has a inlet, but no outlet, either fixedly or transiently. Forexample, when the microfluidic device is made from a gas permeablematerial, such as PDMS, gas present in a dead-end channel can escape, orbe forced out of the channel through the gas permeable material whenurged out by the inflow of liquid through the inlet. This is a preferredexample of blind-filling. Blind-filling can be used with a channel orchamber that has an inlet, and an outlet that is gated or valved by avalve. In this example, blind filling of a gas filled channel or chamberoccurs when the outlet valve is closed while filling the channel orchamber through the inlet. If the inlet also has a valve, that valve canthen be closed after the blind fill is complete, and the outlet can thenbe opened to expose the channel or chamber contents to another channelor chamber. If a third inlet is in communication with the channel orchamber, that third inlet can introduce another fluid, gas or liquid,into the channel or chamber to expel the blind-filled liquid to beexpelled from the channel or chamber in a measured amount. The result issimilar to a sample loop system of an HPLC.

Further Aspects of Retention Mechanisms are Described in Sections V andXIII.

Treatment Mechanisms

Overview

Treatment mechanisms generally comprise any suitable mechanisms forexposing a particle(s) to a reagent(s) and/or a physical condition(s),including fluid-mediated and non-fluid-mediated mechanisms.

Reagents

Particles may be exposed to reagents. A reagent generally comprises anychemical substance(s), compound(s), ion(s), polymer(s), material(s),complex(es), mixture(s), aggregate(s), and/or biological particle(s),among others, that contacts a particle or particle population in amicrofluidic system. Reagents may play a role in particle analysis,including operating as chemical/biological modulators (interactionreagents), detection/assay reagents, solvents, buffers, media, washingsolutions, and/or so on.

Chemical modulators or biological modulators may include any reagentthat is being tested for interaction with particles. Interactiongenerally includes specific binding to particles and/or any detectablegenotypic and/or phenotypic effect on particles (or modulators). Furtheraspects of interactions and genotypic/phenotypic effects that may besuitable are described below in Section XII.

Chemical modulators may include ligands that interact with receptors(e.g., antagonists, agonists, hormones, etc.). Ligands may be smallcompounds, peptides, proteins, carbohydrates, lipids, etc. Furtheraspects of ligands and receptors, and their use in measuringinteraction, or effects on signal transduction pathways, are describedbelow in Section XII.

Alternatively, or in addition, chemical modulators may be nucleic acids.The nucleic acids may be DNA, RNA, peptide nucleic acids, modifiednucleic acids, and/or mixtures thereof, and may be single, double,and/or triple-stranded. The nucleic acids may be produced by chemicalsynthesis, enzymatic synthesis, and/or biosynthesis, and may beplasmids, fragments, sense/antisense expression vectors, reporter genes,vectors for genomic integration/modification (such as targeting nucleicacids/vectors (for knockout/-down/-in)), viral vectors, antisenseoligonucleotides, dsRNA, siRNA, nucleozymes, and/or the like. Nucleicacid reagents may also include transfection reagents to promote uptakeof the nucleic acids by cells, such as lipid reagents (e.g.,lipofectamine), precipitate-forming agents (such as calcium phosphate),DMSO, polyethylene glycol, viral coats that package the nucleic acids,and/or so on.

Modulators may be miscellaneous chemical materials and/or biologicalentities. Miscellaneous chemical modulators may be ions (such ascalcium, sodium, potassium, lithium, hydrogen (pH), chloride, fluoride,iodide, etc.), dissolved gases (NO, CO₂, O₂, etc.), carbohydrates,lipids, organics, polymers, etc. In some embodiments, biologicalmodulators may be exposed to cells, for example, to infect cells, tomeasure cell-cell interactions, etc. Biological modulators may includeany cells, viruses, or organelles, as described above in Section III.

Reagents may be detection/assay reagents. Detection/assay reagentsgenerally comprise any reagents that are contacted with particles tofacilitate processing particles (or particle components) for detectionof a preexisting or newly created aspect of the particles (orcomponents). Detection/assay reagents may include dyes, enzymes,substrates, cofactors, and/or SBP members (see Table 1 of Section VIabove), among others. Dyes, also referred to as labels, generallyinclude any optically detectable reagent. Suitable dyes may beluminophores, fluorophores, chromogens, chromophores, and/or the like.Such dyes may be conjugated to, or may be, SBP members; may act asenzyme substrates; may inherently label cells or cell structures (e.g.,DNA dyes, membrane dyes, trafficking dyes, etc.); may act as indicatordyes (such as calcium indicators, pH indicators, etc.); and/or the like.Enzymes may operate in particle assays by incorporating dyes intoproducts and/or by producing a product that may be detected subsequentlywith dyes, among others. Suitable enzymes may include polymerases (RNAand/or DNA), heat-stable polymerases (such as Taq, VENT, etc.),peroxidases (such as HRP), phosphatases (such as alkaline phosphatase),kinases, methylases, ligases, proteases, galactosidases (such asbeta-galactosidase, glucuronidase, etc.), transferases (such aschloramphenicol acetyltransferase), oxidoreductases (such asluciferase), and/or nucleases (such as DNAses, RNAses, etc.), amongothers. SBP members, such as antibodies, digoxigenin, nucleic acids,etc., may be directly conjugated to dyes, enzymes, and/or other SBPmembers; may be noncovalently bound to dyes and/or enzymes (eitherpre-bound or bound in an additional exposure step); and/or so on.Further aspects of detection/assay reagents, including the types ofassays in which these reagents may be used, are described below inSection XII.

Fluid-Mediated Mechanisms

Treatment mechanisms may use fluid-mediated mechanisms to exposeparticles to reagents. The reagents may be brought to the particles, forexample, when the particles are retained, or the particles may bebrought to the reagents, for example, when the reagents are present (andoptionally retained) in specific portions of fluid networks.

Fluid-mediated mechanisms may be flow-based, field-based, and/orpassive, among others. Flow-based treatment mechanisms may operate byfluid flow, mediated, for example, by gravity flow or active flow(pumping), to carry reagents to particles, or vice versa. In someembodiments, the flow-based treatment mechanisms may operate byregulated transverse (side-to-side) positioning, as describedabove/below in Sections V and XIII, to precisely regulate exposure ofreagents (or particles) to particles (or reagents). By contrast,field-based mechanisms may combine particles and reagents by movingreagents (or particles) with electric fields. The electric fields mayproduce any suitable electrokinetic effects, such as electrophoresis,dielectrophoresis, electroosmosis, etc. Alternatively, or in addition,reagents may be combined with particles by diffusion of the reagents.

Non-Flow-Mediated Mechanisms

Particles in microfluidic systems may be exposed to physicalmodulators/conditions using non-fluid-mediated mechanisms. However,these “non-fluid-mediated” mechanisms may use properties of fluid toassist in their operation, such as transfer of thermal energy orpressure to particles via fluid. The physical modulators/conditions maybe applied to particles from sources that are external and/or internalto the microfluidic systems. Exemplary physical modulators/conditionsmay include thermal energy (heat), radiation (light), radiation(particle), an electric field, a magnetic field, pressure (includingsound), a gravitational field, etc.

Treatment Targets

Treatment mechanisms may act on any suitable particles, including any ofthe particles described above in Section III. The particles may beintact, permeabilized, and/or lysed. Accordingly, treatment mechanismsmay act on released cell components. Particles may be treated in arrays,either serially, for example, using a shared treatment mechanism, and/orin parallel, for example, using distinct and/or shared treatmentmechanisms.

Further aspects of treatment mechanisms are described above in Section V(positioning reagents/fluid/particles) and below in Section XIII.

Measurement Mechanisms

Overview

Particles manipulated by a microfluidic system may be analyzed by one ormore measurement mechanisms at one or more measurement sites. Themeasurement mechanisms generally comprise any suitable apparatus ormethod for detecting a preselected particle or particle characteristic(provided, for example, by the particle, a particle component, and/or anassay product, among others). The measurement sites generally compriseany suitable particle position or positions at which a measurement isperformed, internal and/or external to the system.

Detection Methods

The measurement mechanism may employ any suitable detection method toanalyze a sample, qualitatively and/or quantitatively. Suitabledetection methods may include spectroscopic methods, electrical methods,hydrodynamic methods, imaging methods, and/or biological methods, amongothers, especially those adapted or adaptable to the analysis ofparticles. These methods may involve detection of single or multiplevalues, time-dependent or time-independent (e.g., steady-state orendpoint) values, and/or averaged or (temporally and/or spatially)distributed values, among others. These methods may measure and/oroutput analog and/or digital values.

Spectroscopic methods generally may include detection of any property oflight (or a wavelike particle), particularly properties that are changedvia interaction with a sample. Suitable spectroscopic methods mayinclude absorption, luminescence (including photoluminescence,chemiluminescence, and electrochemiluminescence), magnetic resonance(including nuclear and electron spin resonance), scattering (includinglight scattering, electron scattering, and neutron scattering),diffraction, circular dichroism, and optical rotation, among others.Suitable photoluminescence methods may include fluorescence intensity(FLINT), fluorescence polarization (FP), fluorescence resonance energytransfer (FRET), fluorescence lifetime (FLT), total internal reflectionfluorescence (TIRF), fluorescence correlation spectroscopy (FCS),fluorescence recovery after photobleaching (FRAP), fluorescenceactivated cell sorting (FACS), and their phosphorescence and otheranalogs, among others.

Electrical methods generally may include detection of any electricalparameter. Suitable electrical parameters may include current, voltage,resistance, capacitance, and/or power, among others.

Hydrodynamic methods generally may include detection of interactionsbetween a particle (or a component or derivative thereof) and itsneighbors (e.g., other particles), the solvent (including any matrix),and/or the microfluidic system, among others, and may be used tocharacterize molecular size and/or shape, or to separate a sample intoits components. Suitable hydrodynamic methods may includechromatography, sedimentation, viscometry, and electrophoresis, amongothers.

Imaging methods generally may include detection of spatially distributedsignals, typically for visualizing a sample or its components, includingoptical microscopy and electron microscopy, among others.

Biological methods generally may include detection of some biologicalactivity that is conducted, mediated, and/or influenced by the particle,typically using another method, as described above. Suitable biologicalmethods are described below in detail in Section XII.

Detection Sites

The measurement mechanism may be used to detect particles and/orparticle characteristics at any suitable detection site, internal and/orexternal to the microfluidic system.

Suitable internal detection sites may include any site(s) in or on amicrofluidic system (a chip). These sites may include channels,chambers, and/or traps, and portions thereof. Particles or particlecharacteristics may be detected while the particles (or releasedcomponents/assay products) are stationary or moving. Stationaryparticles may be encountered following particle retention, for example,cells growing in a cell chamber. Moving particles may be encounteredbefore and/or after particle retention, or upon confinement to a region.In particular, particles may be moved past a detection site by anysuitable positioning mechanism, for example, by fluid flow (flow-baseddetection).

Suitable external detection sites may include any site(s) away from orindependent of a microfluidic system. External detection sites may beused to detect a particle or particle characteristic after removal ofparticles (or particle components) from a microfluidic system. Theseexternal sites may be used instead of and/or in addition to internalsites, allowing particles (or particle components) to be furthermanipulated and/or detected. These further manipulations and/ordetection methods may overlap with, but preferably complement, themanipulations and/or methods performed in the microfluidic system,including mass spectrometry, electrophoresis, centrifugation, PCR,introduction into an organism, use in clinical treatment, and/or cellculture, among others.

Detected Characteristics

The measurement method may detect and/or monitor any suitablecharacteristic of a particle, directly and/or indirectly (e.g., via areporter molecule). Suitable characteristics may include particleidentity, number, concentration, position (absolute or relative),composition, structure, sequence, and/or activity among others. Thedetected characteristics may include molecular or supramolecularcharacteristics, such as the presence/absence, concentration,localization, structure/modification, conformation, morphology,activity, number, and/or movement of DNA, RNA, protein, enzyme, lipid,carbohydrate, ions, metabolites, organelles, added reagent (binding),and/or complexes thereof, among others. The detected characteristicsalso may include cellular characteristics, such as any suitable cellulargenotype or phenotype, including morphology, growth, apoptosis,necrosis, lysis, alive/dead, position in the cell cycle, activity of asignaling pathway, differentiation, transcriptional activity, substrateattachment, cell-cell interaction, translational activity, replicationactivity, transformation, heat shock response, motility, spreading,membrane integrity, and/or neurite outgrowth, among others.

Further aspects of detected characteristics and their use in particleassays are described below in Sections XII and XIII.

Release Mechanisms

Overview

A microfluidic system may include any suitable number of particlerelease mechanisms. A release mechanism generally comprises anymechanism(s) for allowing a retained particle to move away from apreselected site/area at which it is retained, including removing,overcoming, and/or rendering ineffective the retention mechanism(s) thatretains the particle. Release mechanisms that are suitable may beselected based, at least partially, on the retaining force. Afterrelease, particles (or particle components) may have any suitabledestination.

Removing the Retaining Force

A release mechanism may operate by removing the retaining force.Accordingly, particles that are retained by a specific mechanism may bereleased by terminating that mechanism. For example, particles retainedby a chemical interaction/bond may be released by cleaving the bond,such as with a protease(s) (e.g., trypsin), or otherwise disrupting theinteraction, such as with altered ionic conditions (e.g., with EDTA) orpH, or with an excess of a SBP member. Similarly, particles retained bya physical barrier, such as a closed valve, may be released bymoving/removing the barrier. Furthermore, particles retained by fluidflow, a vacuum, light, an electrical field, a magnetic field, and/or acentrifugal force may be released by removing/redirecting thecorresponding flow, force, field, etc.

Overcoming the Retaining Force

A release mechanism may operate by overcoming a retaining force with agreater force. Accordingly, particles may be released by any positioningmechanism(s) that applies a force greater than the retaining force. Forexample, retained particles may be released by a releasing flow. Thereleasing flow may be an increased flow rate in the direction of bulkfluid flow, for example, when a particle is weakly retained (such as bygravity/friction, or weak chemical interactions). Alternatively, thereleasing flow may act counter to a retaining flow, for exampleorthogonal or opposite to the retaining flow. For example, the releasingflow may reposition particles to be out of contact with a retainingphysical barrier (see Example 7). Alternatively, or in addition,retained particles may be released by any other suitable positioningmechanism(s), as described above in Section V, that is capable ofgenerating sufficient force.

Rendering Ineffective the Retaining Force

A release mechanism may operate by rendering ineffective the retainingforce on a particle. Such a release mechanism may operate by releasingcomponents of the particle. For example, retained cells may be lysed torelease intracellular components, producing a lysate, or beads may betreated to release associated materials and/or to fragment/disintegratethe beads. Lysis generally includes any partial or complete disruptionof the integrity of a cell-surface membrane, and may be produced viatemperature, a detergent, a ligand, chemical treatment, a change inionic strength, an electric field, etc.

Destination of Released Particles/Components

Released particles and/or particle components may have any suitabledestination(s). Suitable immediate destinations may include apositioning mechanism and/or fluid surrounding the particles. Afterrelease, particles may be repositioned with a positioning mechanism,either nonselectively or selectively. Selective positioning may positionthe particle based on a measured characteristic. Positioning may be to asecond retention mechanism (and/or a culture chamber), to a detectionmechanism (such as a flow-based mechanism), and/or to an outputmechanism. Fluid surrounding the particles may be a suitable destinationfor particle components (such as cells lysates and/or bead components)to be contacted with detection/assay reagents. Alternatively, celllysates and/or bead components may be repositioned as with intactparticles.

Further aspects of release mechanisms and destinations of releasedparticles/components are described below in Section XIII.

Output Mechanisms

Microfluidic systems may include one or more output mechanisms thatinterface with the microfluidic network(s). An output mechanismgenerally comprises any suitable mechanism for outputting material(s)(e.g., fluid, particles, and/or reagents) from a microfluidic system, orportions thereof, including selective and/or bulk mechanisms. The outputmechanism may direct outputted material to any suitable location, suchas an internal and/or external sink. A sink generally comprises anyreceptacle or other site for receiving outputted materials, for disposal(e.g., a waste site) or for further study or manipulation (e.g., acollection site). The outputting of materials from the microfluidicssystem may be facilitated and/or regulated using any suitablefacilitating mechanism, such as sources of internal pressure and/orexternal vacuum. The output mechanism may include a selection mechanism,such as a filter, that selects outputted materials based on somecriterion, such as whether the material is a particle or a fluid.

Cell Culture Mechanisms

Overview

Cells may be cultured using a cell culture mechanism in microfluidicsystems. The cell culture mechanism generally comprises any suitablemechanism for growing cells, including maintenance and/or propagation.Suitable cells are described above in Section III.

Structural Matters

A cell culture mechanism of a microfluidic system may include one ormore culture chambers in which to culture cells. Culture chambers mayhave any suitable size, shape, composition, and/or relationship to otheraspects of microfluidic systems, based on the number of cells to becultured, size of cells, assays to performed on the cells, and/or growthcharacteristics of the cells, among others. The size of a culturechamber may be only large enough to hold one cell, several cells or more(2 to 50), or many cells (50 to 1000 or more) of a given cell size.Accordingly, culture chambers may be defined by a selected portion of apassage, an entire passage, or a set of passages. In some embodiments,culture chambers may be formed by substantially enlarged channels.Culture chambers may have any suitable height that allows cells ofinterest to enter the chamber. This height may be greater than, lessthan, and/or equal to other portions of the microfluidic network. Someor all of the surfaces of a culture chamber, such as the walls, roof,and/or substrate, may be treated or modified to facilitate aspects ofcell culture, particularly specific or nonspecific cell attachment, cellsurvival, cell growth, and/or cell differentiation (or lack thereof),among others. Suitable methods of passage treatment and treatment agentsare described above in Section VI, relative to chemical retentionmechanisms.

Culture Conditions

The cell culture mechanism may culture cells under any suitableenvironmental conditions using any appropriate environmental controlmechanisms. Suitable environmental conditions may include a desired gascomposition, temperature, rate and frequency of media exchange, and/orthe like. Environmental control mechanisms may operate internal and/orexternal to a microfluidic system. Internal mechanisms may includeon-board heaters, gas conduits, and/or media reservoirs. Externalmechanisms may include an atmosphere- and/or temperature-controlledincubator/heat source, and/or a media source external to the system. Anatmosphere-controlled incubator may be more suitable when the system isat least partially formed of a gas-permeable material, such as PDMS.Media, including gas-conditioned media, may be introduced from anexternal source by any suitable input mechanism, including manualpipetting, automated pipetting, noncontact spitting, etc. In someembodiments, the chip may be preincubated with media, which may then bediscarded, prior to the introduction of cells and/or other biologicalmaterials.

Further aspects of cell culture mechanisms, culture chambers, andculture conditions are described below in Example 10, and the materialslisted in Cross-References, particularly R. Ian Freshney, Culture ofAnimal Cells: A Manual of Basic Technique (4^(th) ed. 2000), which isincorporated herein by reference.

Particle-Based Manipulations

Overview

Microfluidic systems are used for particle manipulations. Particlemanipulations generally comprise any suitable sequence of unitaryoperations, for performing a desired function or assay. Unitaryoperations may be performed by each of the mechanisms described above inSections IV to X, among others.

Exemplary Sequences of Operations

FIG. 1 shows an exemplary method 100 for microfluidic manipulation andanalysis of particles with systems of the invention. Each step of method100 may be repeated any suitable number of times and in any appropriateorder, as described below, based on the application. Exemplary sequencesof steps are indicated by arrows.

Particles typically are initially inputted in an input step, shown at101. Particle input introduces particles to a microfluidic system andmay be mediated by any of the input mechanisms described above inSection IV.

Particles next are typically positioned, shown at 102. Positioning movesparticles to selected positions along passages (longitudinalpositioning), and/or to selected positions along one or more axesgenerally orthogonal to the long axis (transverse positioning). Suitablepositioning mechanisms that mediate one or both of these particlemovements are described above in Section V.

Particle positioning may lead to one of two paths, shown at 103 and 104.Path 103 leads to particle output, shown at 105. Particle output may bemediated by one of the output mechanisms described above in Section X,and may be used to discard, collect, and/or transfer particles forfurther analysis, among others. Path 104 leads to one or more of threeoperations, particle retention 106, particle treatment 107, and/orparticle measurement/detection 108. These operations may be conducted inany suitable order, for any desired number of times. Particle retentionmechanisms, treatment mechanisms, and measurement mechanisms aredescribed above in Sections VI, VII, and VIII, respectively.

The steps of treating and/or measuring particles may be carried out withor without particle retention. Accordingly, the steps of treating and/ormeasuring particles may be followed directly by additional positioning102, or first may use a release step, shown at 109, if particles havebeen retained. Suitable release mechanisms are described above inSection IX. Alternatively, microfluidic systems may be discarded beforeparticle release, additional positioning, and/or output.

Particles that have returned to the positioning step after entering path104 may be manipulated further. Some or all of these particles may berepositioned to path 103 to be outputted 105. Alternatively, or inaddition, some or all of these particles may be directed back to path104 to be further treated, retained, and/or measured. Therefore, method100 enables any suitable sequence of particle manipulations and analysesat one or plural positions within a microfluidic system.

Exemplary sequences of operations may be illustrated further as follows.For the following discussion, the operations performed by the steps ofmethod 100 are abbreviated with the following single underlined letters:Input, Position, Retain, Treat, Measure, rElease, and Output.

A basic manipulation of microfluidic analyses is IP. This sequence ofsteps may lead to output (IPO) or to (path 104), resulting in the basicretention sequence IPR, flow-based measurement, IPM, or flow-basedtreatment, IPT.

Retained particles may be subjected to any suitable additional steps.The particles may be treated (IPRT), measured (IPRM), repeatedlymeasured over time (IPRMMM . . . ), treated and then measured (IPRTM),or repeatedly treated and measured (IPRTMTMTM . . . ). Retainedparticles may be released (IPR . . . E) after optional treatment and/ormeasurement. Released particles may be repositioned and then outputted(IPR . . . EPO); measured during flow (IPR . . . EPM); treated (IPR . .. EPT); treated and measured (IPR . . . EPTM); retained and treated (IPR. . . EPRT); retained, treated, and measured, (IPR . . . EOPRTM); and/orso on.

Cell-Based Assays/Methods

The microfluidic systems of the invention may be used for any suitablecell assays or methods, including any combinations of cells, cellselection(s) (by selective retention), treatment(s), and/ormeasurement(s), as described above in Sections III, VI, VII, and VIII,respectively.

The cell assays may characterize cells, either with or without additionof a modulator. Cell assays may measure cell genotypes, phenotypes,and/or interactions with modulators. These assays may characterizeindividual cells and/or cell populations/groups of any suitable size.Cells may be characterized in the absence of an added modulator todefine one or more characteristics of the cells themselves.Alternatively, or in addition, cell may be characterized in the presenceof an added modulator to measure interaction(s) between the cells andthe modulator. Moreover, cells may be exposed to a selectedconcentration of a reagent, or a plurality of concentrations of areagent. In other embodiments, cells are exposed to a gradient ofconcentrations of reagent to determine whether such cells will beattracted or repelled by increasing amounts of such reagent.

In other embodiments, a quantity of cells may be measured out by firstfilling a measuring chamber having at least one inlet, the inlet havingat least one valve, where the valve is opened, cells are introduced intothe chamber, preferably by blind filling a dead-end chamber, or byopening up an outlet valve to an outlet in communication with thechamber, the outlet having a retention mechanism for preventing thecells from exiting the chamber. The measure amount of cells is thendisplaced to a culturing region for culturing.

In other embodiments, a first type of cell is grown in fluidcommunication with a second type of cell, wherein the first type of cellis affected by the presence of the second type of cell, preferably as aco-culture or feeder type relationship. The cells of the first type andthe cells of the second type are kept separate from each other by aretention mechanism, although fluid, preferably liquid, is permitted tobe in joint contact with each type of cell so that sub-cellular orbiochemical materials may be exchanged between cell types.

Genotypic Assays

Genotypic assays may be conducted on cells in microfluidic systems tomeasure the genetic constitution of cells. The genotypic assays may beconducted on any suitable cell or cell populations, for example, patientsamples, prenatal samples (such as embryonic, fetal, chorionic villi,etc.), experimentally manipulated cells (such as transgenic cells),and/or so on. Such genotypic aspects may include copy number (such asduplication, deletion, amplification, and/or the like) and/or structure(such as rearrangement, fusion, number of repeats (such as dinucleotide,triplet repeats, telomeric repeats, etc.), mutation, gene/pseudogene,specific allele, presence/absence/identity/frequency of singlenucleotide polymorphisms, integration site, chromosomallepisomal, and/orthe like) of a nuclear and/or mitochondrial gene(s), genomic region(s),and/or chromosomal region (s) (such as telomeres, centromeres,repetitive sequences, etc.). Methods for genotypic assays may includenucleic acid hybridization in situ (on intact cells/nuclei) or with DNAreleased from cells, for example, by lysing the cells. Nucleic acidhybridization with nucleic acids may be carried out with a dye-labeledprobe, a probe labeled with a specific binding pair (see Section VI), astem-loop probe carrying an energy transfer pair (such as a “molecularbeacon”), and/or with a probe that is labeled enzymatically afterhybridization (such as by primer extension with a polymerase,modification with terminal transferase, etc). Alternatively, or inaddition, methods for genotypic assays may include polymerase-mediatedamplification of nucleic acids, for example, by thermal cycling (PCR) orby isothermal strand-displacement methods. In some embodiments,genotypic assays may use electrophoresis to assist in analysis ofnucleic acids. Related gene-based assays may measure other aspects ofgene regions, genes, chromosomal regions, whole chromosomes, or genomes,using similar assay methods, and suitable probes or DNA dyes (such aspropidium iodide, Hoechst, etc.). These other aspects may include totalDNA content (for example 2N, 4N, 8N, etc., to measure diploid,tetraploid, or polyploid genotypes and/or cell cycle distribution),number or position of specific chromosomes, and/or position of specificgenes (such as adjacent the nuclear membrane, another nuclear structure,and so on).

Phenotypic Assays

Phenotypic assays may be conducted to characterize cells in microfluidicsystems, based on genetic makeup and/or environmental influences, suchas presence of modulators. These assays may measure any molecular orcellular aspect of whole cells, cellular organelles, and/or endogenous(native) or exogenous (foreign) cell constituents/components.

Aspects of a whole cell or whole cell population may include number,size, density, shape, differentiation state, spreading, motility,translational activity, transcriptional activity, mitotic activity,replicational activity, transformation, status of one or more signalingpathways, presence/absence of processes, intact/lysed, live/dead,frequency/extent of apoptosis or necrosis, presence/absence/efficiencyof attachment to a substrate (or to a passage), growth rate, cell cycledistribution, ability to repair DNA, response to heat shock, natureand/or frequency of cell-cell contacts, etc.

Aspects of cell organelles may include number, size, shape,distribution, activity, etc. of a cell's (or cell population's) nuclei,cell-surface membrane, lysosomes, mitochondria, Golgi apparatus,endoplasmic reticulum, peroxisomes, nuclear membrane, endosomes,secretory granules, cytoskeleton, axons, and/or neurites, among others.

Aspects of cell constituents/components may include presence/absence orlevel, localization, movement, activity, modification, structure, etc.of any nucleic acid(s), polypeptide(s), carbohydrate(s), lipid(s),ion(s), small molecule, hormone, metabolite, and/or a complex(es)thereof, among others. Presence/absence or level may be measuredrelative to other cells or cell populations, for example, with andwithout modulator. Localization may be relative to the whole cell orindividual cell organelles or components. For example, localization maybe cytoplasmic, nuclear, membrane-associated, cell-surface-associated,extracellular, mitochondrial, endosomal, lysosomal, peroxisomal, and/orso on. Exemplary cytoplasmic/nuclear localization may includetranscription factors that translocate between these two locations, suchas NF-κB, NFAT, steroid receptors, nuclear hormone receptors, and/orSTATs, among others. Movement may include intracellular trafficking,such as protein targeting to specific organelles, endocytosis,exocytosis, recycling, etc. Exemplary movements may include endocytosisof cell-surface receptors or associated proteins (such as GPCRs,receptor tyrosine kinases, arrestin, and/or the like), eitherconstitutively or in response to ligand binding. Activity may includefunctional or optical activity, such as enzyme activity, fluorescence,and/or the like, for example, mediated by kinases, phosphatases,methylases, demethylases, proteases, nucleases, lipases, reporterproteins (for example beta-galactosidase, chloramphenicolacetyltransferase, luciferase, glucuronidase, green fluorescent protein(and related derivatives), etc.), and/or so on. Modification may includethe presence/absence, position, and/or level of any suitable covalentlyattached moiety. Such modifications may include phosphorylation,methylation, ubiquitination, carboxylation, and/or farnesylation, amongothers. Structure may include primary structure, for example afterprocessing (such as cleavage or ligation), secondary structure ortertiary structure (e.g., conformation), and/or quaternary structure(such as association with partners in, on, or about cells). Methods formeasuring modifications and/or structure may include specific bindingagents (such as antibodies, etc.), in vivo or in vitro incorporation oflabeled reagents, energy transfer measurements (such as FRET), surfaceplasmon resonance, and/or enzyme fragment complementation or two-hydridassays, among others.

Nucleic acids may include genomic DNA, mitochondrial DNA, viral DNA,bacterial DNA, phage DNA, synthetic DNA, transfected DNA, reporter geneDNA, etc. Alternatively, or in addition, nucleic acids may include totalRNAs, hnRNAs, mRNAs, tRNAs, siRNAs, dsRNAs, snRNAs, ribozymes,structural RNAs, viral RNAs, bacterial RNAs, gene-specific RNAs,reporter RNAs (expressed from reporter genes), and/or the like. Methodsfor assaying nucleic acids may include any of the techniques listedabove under genotypic assays. In addition, methods for assaying nucleicacids may include ribonuclease protection assays.

Polypeptides may include any proteins, peptides, glycoproteins,proteolipids, etc. Exemplary polypeptides include receptors, ligands,enzymes, transcription factors, transcription cofactors, ribosomalcomponents, regulatory proteins, cytoskeletal proteins, structuralproteins, channels, transporters, reporter proteins (such as thoselisted above which are expressed from reporter genes), and/or the like.Methods for measuring polypeptides may include enzymatic assays and/oruse of specific binding members (such as antibodies, lectins, etc.),among others. Specific binding members are described in Section VI.

Carbohydrates, lipids, ions, small molecules, and/or hormones mayinclude any compounds, polymers, or complexes. For example,carbohydrates may include simple sugars, di- and polysaccharides,glycolipids, glycoproteins, proteoglycans, etc. Lipids may includecholesterol and/or inositol lipids (e.g., phosphoinositides), amongothers; ions may include calcium, sodium, chloride, potassium, iron,zinc, hydrogen, magnesium, heavy metals, and/or manganese, among other;small molecules and/or hormones may include metabolites, and/or secondmessengers (such as cAMP or cGMP, among others), and/or the like.Concentration gradients and/or movement of ions may provide electricalmeasurements, for example, by patch-clamp analysis, as described inExamples 11 and 12.

Interaction Assays

Interaction generally comprises any specific binding of a modulator to acell or population of cells, or any detectable change in a cellcharacteristic in response to the modulator. Specific binding is anybinding that is predominantly to a given partner(s) that is in, on, orabout the cell(s). Specific binding may have a binding coefficient withthe given partner of about 10⁻³ M and lower, with preferred specificbinding coefficients of about 10⁻⁴ M, 10⁻⁶ M, or 10⁻⁸ M and lower.Alternatively, interaction may be any change in a phenotypic orgenotypic characteristic, as described above, in response to themodulator.

Interaction assays may be performed using any suitable measurementmethod. For example, the modulator may be labeled, such as with anoptically detectable dye, and may be labeled secondarily afterinteraction with cells. Binding of the dye to the cell or cells thus maybe quantified. Alternatively, or in addition, the cell may be treated orotherwise processed to enable measurement of a phenotypic characteristicproduced by modulator contact, as detailed above and in Section VIII.

Cells and/or cell populations may be screened with libraries ofmodulators to identify interacting modulators and/or modulators withdesired interaction capabilities, such as a desired phenotypic effect(such as reporter gene response, change in expression level of a nativegene/protein, electrophysiological effect, etc.) and/or coefficient ofbinding. A library generally comprises a set of two or more members(modulators) that share a common characteristic, such as structure orfunction. Accordingly, a library may include two or more smallmolecules, two or more nucleic acids, two or more viruses, two or morephages, two or more different types of cells, two or more peptides,and/or two or more proteins, among others.

Signal Transduction Assays

Microfluidic assays of cells and/or populations may measure activity ofsignal transduction pathways. The activity may be measured relative toan arbitrary level of activity, relative to other cells and/orpopulations (see below), and/or as a measure of modulator interactionwith cells (see above).

Signal transduction pathways generally comprise any flow of informationin a cell. In many cases, signal transduction pathways transferextracellular information, in the form of a ligand(s) or othermodulator(s), through the membrane, to produce an intracellular signal.The extracellular information may act, at least partially, by triggeringevents at or near the membrane by binding to a cell-surface receptor,such as a G Protein-Coupled Receptor (GPCR), a channel-coupled receptor,a receptor tyrosine kinase, a receptor serine/threonine kinase, and/or areceptor phosphatase, among others. These events may include changes inchannel activity, receptor clustering, receptor endocytosis, receptorenzyme activity (e.g., kinase activity), and/or second messengerproduction (e.g., cAMP, cGMP, diacylglcyerol, phosphatidylinositol,etc.). Such events may lead to a cascade of regulatory events, such asphosphorylation/dephosphorylation, complex formation, degradation,and/or so on, which may result, ultimately, in altered gene expression.In other cases, modulators pass through the membrane and directly bindto intracellular receptors, for example with nuclear receptors (such assteroid receptors (GR, ER, PR, MR, etc.), retinoid receptors, retinoid Xreceptor (RXRs), thyroid hormone receptors, peroxisomeproliferation-activating receptors (PPARs), and/or xenobiotic receptors,among others). Therefore, any suitable aspect of this flow ofinformation may be measured to monitor a particular signal transductionpathway.

The activity measured may be based at least partially, on the type ofsignal transduction pathway being assayed. Accordingly, signaltransduction assays may measure ligand binding; receptorinternalization; changes in membrane currents; association of receptorwith another factor, such as arrestin, a small G-like protein such asrac, or rho, and/or the like; calcium levels; activity of a kinase, suchas protein kinase A, protein kinase C, CaM kinase, myosin light chainkinase, cyclin dependent kinases, PI3-kinase, etc.; cAMP levels;phosholipase C activity; subcellular distribution of proteins, forexample, NF-κB, nuclear receptors, and/or STATs, among others.Alternatively, or in addition, signal transduction assays may measureexpression of native target genes and/or foreign reporter genes thatreport activity of a signal transduction pathway(s). Expression may bemeasured as absence/presence or level of RNA, protein, metabolite, orenzyme activity, among others, as described above.

Comparison of Cells and/or Cell Populations

Cell-based assays may be used to compare genotypic, phenotypic, and/ormodulator interaction of cells and/or populations of cells. The cellsand/or populations may be compared in distinct microfluidic systems orwithin the same microfluidic system. Comparison in the same microfluidicsystem may be conducted in parallel using a side-by-side configuration,as exemplified by Example 3, in parallel at isolated sites, asexemplified by Example 4, and/or in series, as exemplified by Example 5.

Single-Cell Assays

Microfluidic systems may be used to perform single-cell assays, whichgenerally comprise any assays that are preferably or necessarilyperformed on one cell at a time. Examples of single cell assays includepatch-clamp analysis, single-cell PCR, single-cell fluorescence in situhybridization (FISH), subcellular distribution of a protein, and/ordifferentiation assays (conversion to distinct cell types). In somecases, single-cell assays may be performed on a retained group of two ormore cells, by measuring an individual characteristic of one member ofthe group. In other cases, single-cell assays may require retention of asingle cell, for example, when the cell is lysed before the assay.

Sorting/Selection

Microfluidic systems may be used to sort or select single cells and/orcell populations. The sorted/selected cells or populations may beselected by stochastic mechanisms (see Example 2), size, density,magnetic properties, cell-surface properties (that is, ability to adhereto a substrate), growth and/or survival capabilities, and/or based on ameasured characteristic of the cells or populations (such as response toa ligand, specific phenotype, and/or the like). Cells and/or populationsmay be sorted more than once during manipulation and/or analysis in amicrofluidic system. In particular, heterogeneous populations of cells,such as blood samples or clinical biopsies, partially transfected ordifferentiated cell populations, disaggregated tissues, natural samples,forensic samples, etc. may be sorted/selected. Additional aspects ofcell sorting and suitable cells and cell populations are described abovein Section III and below in Examples 9, 15, 23, and 26.

Storage/Maintenance

Microfluidic systems may perform storage and/or maintenance functionsfor cells. Accordingly, cells may be introduced into micro fluidicsystems and cultured for prolonged periods of time, such as longer thanone week, one month, three months, and/or one year. Using microfluidicsystems for storage and/or maintenance of cells may consume smalleramounts of media and space, and may maintain cells in a more viablestate than other storage/maintenance methods. Additional aspects ofstoring and maintaining cells in microfluidic systems are included inSection XI above and Example 10 below.

Assays/Methods with Other Particles

Microfluidic systems may be used for any suitable virally based,organelle-based, bead-based, and/or vesicle-based assays and/or methods.These assays may measure binding (or effects) of modulators (compounds,mixtures, polymers, biomolecules, cells, etc.) to one or more materials(compounds, polymers, mixtures, cells, etc.) present in/on, orassociated with, any of these other particles. Alternatively, or inaddition, these assays may measure changes in activity (e.g., enzymeactivity), an optical property (e.g., chemiluminescence, fluorescence,or absorbance, among others), and/or a conformational change induced byinteraction.

In some embodiments, beads may include detectable codes. Such codes maybe imparted by one or more materials having detectable properties, suchas optical properties (e.g., spectrum, intensity, and or degree offluorescence excitation/emission, absorbance, reflectance, refractiveindex, etc.). The one or more materials may provide nonspatialinformation or may have discrete spatial positions that contribute tocoding aspects of each code. The codes may allow distinct samples, suchas cells, compounds, proteins, and/or the like, to be associated withbeads having distinct codes. The distinct samples may then be combined,assayed together, and identified by reading the code on each bead.Suitable assays for cell-associated beads may include any of the cellassays described above.

Suitable protocols for performing some of the assays described in thissection are included in Joe Sambrook and David Russell, MolecularCloning: A Laboratory Manual (3^(rd) ed. 2000), which is incorporatedherein by reference.

Examples

The following examples describe selected aspects and embodiments of theinvention, including methods of fabricating, integrating, and usingmicrofluidic systems, and devices, and mechanisms for manipulation andanalysis of particles. These examples are included for illustration andare not intended to limit or define the entire scope of the invention.

Many of the examples presented below include figures showing molds,fluid layers, and/or control layers that are color-coded. Since moldsand fluid or control layers have complementary patterns, the color-codedschemes generally represent both molds and fluid or control layers,although one or the other is often designated in the correspondingdescription. Throughout these examples, the colors of molds and/orfluidic layers have the following meanings: regions in red have a heightof approximately 20 μm, and a rectangular cross-sectional geometry;regions in blue have a height of about 20 μm, and asemi-circular/arcuate cross-sectional geometry; regions in turquoisehave a height of about 5 μm and a rectangular cross-sectional geometry;and regions in white are not raised from the general surface of the moldand/or form a portion of the substrate-contacting surface of a fluidlayer. The widths of these regions are generally cited in the text.

Dimensions and cross-sectional geometries presented in these examplesare exemplary only, being designed for particles of about 8 to 12 μm indiameter. Accordingly, any absolute or relative dimensions orcross-sectional geometries may be selected based the application and thesize of input particles being analyzed. Thus, the regions in red andblue may have a height of about 0.5 to 100, 1 to 75, or 2 to 50 μm.Regions in turquoise may have a height of about 0.1 to 50, 0.2 to 25, orabout 0.5 to 20 μm. In addition, these regions may have any suitablecross-sectional geometries based on the application. Furthermore,regions in red and blue may have any suitable width based on theirfunction. For example, regions in red used for particle positioning mayhave widths of at least about 2, 10, 20, or 50 μm. By contrast, regionsin red used for reagent dispensing may have smaller widths of at leastabout 0.2, 1, 2, or 5 μm. Regions in blue may have widths of at leastabout 5, 10, 20, or 50 μm.

Example 1 Cell Positioning and Retention Mechanisms

This example describes microfluidic systems for positioning and/orretaining single particles or groups of particles, based, at least inpart, on divergent flow paths; see FIGS. 2-4.

Background

There are many cell analyses that benefit from or require the precisepositioning and retention of a single cell or a small group of cells. Inparticular, positioned and retained cells may be treated and observed inreal time. However, currently available mechanisms for positioning andretaining cells are either expensive and labor intensive, or impreciseand deleterious to cells. For example, micromanipulators enable a userto select and precisely position a single cell. However,micromanipulators are expensive, and require that users observe the cellthroughout the micromanipulation. Hence, the user can only position onecell at a time. At the other extreme, filters offer a crude, but muchcheaper and faster mechanism for positioning and retaining cells.However, filters have a number of disadvantages. For example, they areeasy to clog, difficult to control (particularly with regard to thenumber of retained cells), and potentially harmful to particles such ascells due to the pressure drop across the filter. Therefore, there is aneed for cell positioning and retention systems that are economical,guided automatically without optical monitoring, and/or able to gentlymanipulate cells without substantially damaging them.

Description

This example describes mechanisms for positioning and/or retainingparticles such as cells and/or beads without requiring opticalmonitoring. Once retained, the particles may be analyzed by any suitablemethod, including optical and electrical methods, among others. Thedescribed mechanisms use a microfluidic flow path that diverges to forma quasi-stagnant fluidic region at the position of divergence. Particlesentering this quasi-stagnant fluidic region from a microfluidic streamexperience a reduction in velocity, which may be exploited to effecttheir “soft landing” in a suitable retention structure or trap.Accordingly, the retained particles are more likely to be undamaged andsuitable for subsequent analyses.

Embodiment 1

FIG. 2A shows a system 110 for microfluidic manipulation and/or analysisof particles, in accordance with aspects of the invention. System 110includes (1) an input reservoir 112, (2) a microfluidic network 114having three fluidic channels 116, 118, 120, and (3) two output or wastereservoirs 122, 124. Particles are loaded, generally in suspension, intoinput reservoir 112. The loaded particles may enter network 114 inresponse to net fluid flow, shown as flow streams 126, 128, 130, betweenthe input and waste reservoirs. The net fluid flow may be determined byactive and/or passive flow, mediated, for example, by pumping and/orgravity, respectively.

The bifurcation of fluid flow stream 126 into flow streams 128, 130creates a positioning mechanism 132. This positioning mechanism uses areduced-velocity flow stream 134, shown as a dotted arrow, to gentlyposition a fraction of particles through an extension of flow stream126.

Particles may be carried by flow stream 134 into a suitable retentionmechanism 136. In system 110, this retention mechanism includes a recess138 formed in opposing wall 140, near a terminal end of reduced-velocityflow stream 134. Recess 138 may have a width and depth that accommodatesone particle or a group of two or more particles. Recess 138 includesretention structures 142 that block movement of retained particles,generally in the direction of flow streams 128, 130. The depth of recess138, coupled with any extension of retention structures 142, generallyaway from wall 140, may determine the number of particles retained andtheir associated retention efficiency. Thus, retention mechanism 136 mayeffect stable or transient retention of particles. Transient retentionmay provide an average time of occupancy that is suitable for treatmentand/or analysis, followed by stochastic loss and replacement of aparticle or particles by other particles entering along reduced-velocityflow stream 134.

Particles retained by retention mechanism 136 may be treated and/oranalyzed. In some embodiments, retained particles are analyzedelectrically, for example, using an electrode 143. Alternatively, or inaddition, retained particles may be treated and/or analyzed and thenremoved by a suitable release mechanism 144. For example, in system 110,the release mechanism applies a dislodging pressure on retained cellsthat opposes flow stream 134. Release mechanisms are described furtherin Section IX above and in Examples 7 and 26 below.

Embodiment 2

FIG. 2B shows another system 110′ for microfluidic manipulation and/oranalysis of particles, in accordance with aspects of the invention. Theoperational principles for system 110′ of FIG. 2B are similar to thosefor system 110 of FIG. 2A. However, channels 118′ and 120′ diverge lessthan 90° from channel 116′ in system 110′, in contrast to theirorthogonally directed counterparts in system 110. Consequently, agreater fraction of particles may be positioned in flow stream 134′ insystem 110′ than in flow stream 134 in system 110, but a greaterdislodging force also may be present. In other embodiments, the outputchannels may have any suitable angles of divergence, including greaterthan 90°, and/or they may have unequal angles of divergence. The anglesof divergence and any asymmetry in the two fluid paths may be alterableto select the number of particles trapped and/or retained and theirpositions within the trap.

Embodiment 3

FIG. 3 shows yet another system 170 for microfluidic manipulation and/oranalysis of particles, in accordance with aspects of the invention.System 170 includes (1) a fluidic network 172 of channels 174, 176, 178and (2) a retention mechanism or trap 180. A flow stream 181 bringsinput sample and fluid to a T-junction 182, at which stream 181 isdivided into orthogonally directed, primary flow-streams 184, 186. As insystems 110, 110′ of FIGS. 2A and 2B, a reduced velocity, positioningflow-stream 188 extends from stream 181, between primary streams 184,186, toward opposing wall 188. However, unlike systems 110 and 110′,system 170 also includes partitions 192, 194 (“P” and “Q”, respectively)in the form of rectangular blocks. Partitions 192, 194 divide the mainchannels to create secondary channels 196, 198, which extend generallyparallel to main channels 176, 178. These secondary channels dividepositioning flow-stream 188 and direct it orthogonally in oppositedirections, as shown by secondary flow streams 200, 202. Secondary flowstreams transport fluid at a lower velocity than primary streams 184,186 because of their position within network 172.

FIG. 4 shows system 170 during particle input, after positioning andretention of a single particle 204 between partitions 192, 194 by trap180. Particles 206 entering network 172 may travel along flow stream181, generally in both central and lateral positions within channel 174.Laterally positioned cells, such as cells 208, follow primary flowstreams 184, 186 along channels 176, 178. In contrast, centrallypositioned cells, such as cells 210, follow positioning stream 188toward a slot or gap 212 between partitions 192, 194. In thisembodiment, gap 212 is slightly wider than the diameter of cells 206, sothat it will accept only one cell. In other embodiments, and/or forother cells, gap 212 may be wide enough to accept two or more cells.Whatever the width of gap 212, wall 190 and partitions 192, 194, form aretention site 214 at which cell 204 or cells may be stably retained.Once cell 204 is positioned at the retention site by trap 180, itspresence may tend to block or diminish fluid flow along secondarystreams 200, 202, through secondary channels 196, 198 (see FIG. 3).Accordingly, secondary streams 200, 202 have diminished capacity to drawadditional cells between partitions 192, 194. As a result, in someembodiments, trap 180 may preferentially retain only one cellautomatically, without any need for optical monitoring duringpositioning and/or retention. Thus, retention site 214 may bedimensioned based on the size of cells to be retained. For example,eukaryotic cells typically are about 2 to 10 μm in diameter, so gap 212may be slightly wider than this diameter, whereas secondary channels196, 198 may be slightly narrower than this diameter, to prevent entryof cells into these channels.

Retained cell 204 may be treated and/or analyzed using any suitablemethod, such as optical and/or electrical detection of cellcharacteristics, as described above in Section VIII. This treatmentand/or analysis may be facilitated by a microchannel 216 that extendsoutward from wall 190 into chamber 218. Microchannel 216 is smaller thanthe diameter of retained cell 204 and may be used to exert positiveand/or negative pressure on the retained cell, or apply and/or measurean electrical potential and/or current across the retained cell, amongothers, as described below in Examples 11 and 12.

Example 2 Microfluidic Systems for Trapping and Perfusing Particles

This example describes microfluidic systems that position and retainsingle particles or sets of particles, and allow rapid, preciseperfusion of the retained particles or sets of particles with reagents;see FIGS. 5-11C.

Background

Many cell studies benefit from analysis of a population of cells. Thepopulation may provide discrete information from individual cells of thepopulation and averaged information from the entire population.Accordingly, a population of cells may allow concurrent analysis ofdistinct types of cells when the population is heterogeneous, or a rangeof cell phenotypes or responses when the population is homogeneous orclonal. Therefore, studies of cells in a microfluidic environment wouldbenefit from microfluidic systems that automatically position and/orretain a set of cells at a preselected site on a microfluidic chip.Furthermore, these studies would benefit from mechanisms that allow theretained set of cells to be perfused with selected reagents, such asdrugs, test compounds, or labels, in a controllable and definablemanner.

Description

This example describes microfluidic systems that enable a user to trapmultiple cells within a cell retention chamber, and perfuse the trappedcells with reagents for controlled intervals. These systems may beformed by any suitable method, including multilayer soft lithographyinvolving multiple layers of photoresist, for example, using moldsfabricated as described below in Example 13 and elsewhere in thisDetailed Description, and in the patent applications listed above underCross-References and incorporated herein by reference. Accordingly, insome embodiments, the cross-sectional geometry of fluidic channels mayvary between rectangular in flow channels and arcuate at the position ofvalves.

Embodiment 1

FIGS. 5-11 show a system 250 for microfluidic analysis of cellpopulations. This system is described in detail below, including (a)system description, (b) system production, (c) system operation, and (d)system protocols.

System Description

FIG. 5 shows a portion of a system 250 for microfluidic analysis of cellpopulations. System 250 includes a microfluidic layer 252 and a controllayer 254. Microfluidic layer 252 forms a microfluidic network 256 ofinterconnected channels, depicted in blue and orange. Control layer 254is positioned over, and abutting, the microfluidic layer, and includesvalves and pumps (see also FIG. 8), depicted in purple. Exemplarydimensions presented below for system 250 are based on cell diameters ofabout 8 to 12 μm.

The microfluidic layer includes microfluidic channels with distinctgeometries and functions. Blue, flow channels 258 have a semi-circularor arcuate cross-sectional profile and are positioned generally upstreamand downstream of mechanisms for cell positioning, retention, and/ortreatment, which are described below. These flow channels havecross-sectional profiles that allow the channels to be acted uponeffectively by valves and pumps present in control layer 254. In thisexample, flow channels are about 200 μm wide and 20 μm high. Incontrast, orange, cell channels 260 have a rectangular profile. In thisexample, cell channels are about 100 μm wide and 20 μm high. Becausechannel height does not restrict lateral movement, at least to firstorder, the cells or particles can travel freely within the cell channel,following the walls or more central positions based on the particularlaminar flow stream that carries a particular cell or particle. Thus,these cell channels are used to position cells to preselected laminarflow streams and preselected regions of the microfluidic network.Perfusion channels 262, described more fully below, also are shown inorange and function to controllably perfuse retained cells. In thisparticular example, perfusion channels are about 10 μm wide.

System 250 includes an input mechanism 263, a positioning mechanism 264,a retention mechanism 266, and a perfusion mechanism 268. Thepositioning and retention mechanisms function together to position andtrap cells in a retention or capture chamber 270. The perfusionmechanism functions to effect delivery of reagents to the cells inretention chamber 270, typically after cell retention.

Input mechanism 263 introduces particles into the system, using an inputreservoir or well, as described below (see FIG. 8).

Positioning mechanism 264 operates to increase the probability thatinput cells will enter the retention chamber. Mechanism 264 operatesthrough convergent flow streams that join but remain segregated in alaminar distribution. Input flow streams 272, 274, 276 carry fluid alongflow channels 278, 280, 282, respectively. However, channel 280 also maycarry cells, whereas flanking channels 278, 282 generally do not. As aresult, at confluence 284, flow stream 274 occupies a central portion,flanked by flow streams 272, 276. Accordingly, the accompanying cellsare focused to a central portion of combined stream 286. In someembodiments, additional flow streams may be included, and/or cells maybe included in other flow streams, as exemplified below in Example 3.

FIGS. 6 and 7 show, respectively, corresponding actual and schematicviews of the retention mechanism or trap 266 of FIG. 5. The retentionmechanism includes a partially closed retention or capture chamber 270.Chamber 270 may have a size of about 60-100 μm long, 50-100 μm wide, and20 μm high. Chamber 270 is formed by opposing channel wall 288, frontwall 290, side walls 292, 294, and top and bottom walls (not shown).Front wall includes an aperture 296 through which cells enter thechamber from a reduced-velocity stream 298, extending from combinedstream 286. The reduced-velocity stream may be less damaging to cellsthat enter the chamber, increasing viability and the probability of afruitful analysis. Aperture 296 is about 5-20 μm wide and may have aheight corresponding to some or all of the channel height. Fluidentering aperture 296 as part of stream 298 may pass through side-wallchannels 300. In this example, each side wall includes three side-wallchannels 300, which have a rectangular profile about 10 μm wide and 5 μmhigh. In general, the side-wall channels are dimensioned to selectivelyretain cells or particles of interest, while allowing fluid or smallercells or particles to pass through. Thus, chamber 270 functions as afilter. However, in contrast to standard filters, only a fraction ofinput cells enter chamber 270. The fraction may be less than about 1 in10, 1 in 100 or 1 in 1,000, among others, depending on the design of theretention chamber, the speed of the input fluid stream, and the size anddensity of particles, among others.

FIG. 7 shows a focused stream of cells 302 flowing toward chamber 270.Cells 302 either enter aperture 296 or are carried orthogonally bychannels 304, 306. Within chamber 270 microstreams 308 connect chamber270 with side-wall channels 300.

Perfusion mechanism 268 provides precisely controlled exposure toreagents for trapped cells in chamber 270. FIG. 5 shows the generaldesign of the perfusion mechanism. Trapped cells are selectively exposedto buffer or reagent streams carried by one of two or more perfusionchannels 310, 312. Fluid, such as media, buffer, and/or reagent, flowsthrough perfusion channels 310 and/or 312 and joins focusing bufferstream 314. During perfusion, focusing buffer stream 314 is produced byinput fluid from one or more input reservoirs “B,” described more fullybelow, flowing past chamber 270 in a single stream. Thus, the stream nois longer split as occurs during cell positioning and retention, asshown in FIG. 7. Due to laminar flow and the position of perfusionchannels 310, 312, fluid from either one of these channels enters tojoin main flow stream 314 on the side of the main flow stream nearestchamber 270. Therefore, the trapped cells are exposed to fluid fromperfusion channel 310 or 312. However, if fluid is flowing from bothperfusion channels, fluid from perfusion channel 312 shields trappedcells from fluid flowing from perfusion channel 310, such as a reagent.Accordingly, the contents of perfusion channel 312 may be referred to asa shield liquid or shield buffer. With concurrent flow from bothperfusion channels, cells may be rapidly exposed to a reagent fromperfusion channel 310 by stopping flow from channel 312. Stopping theflow of the perfusion buffer may expose cells to reagent within a veryshort time, in some cases about 150 msec after stopping flow. Therefore,cell analyses that require precise control of reagent exposure tomeasure rapid cell responses may be conducted reproducibly with therapid response times afforded by this microfluidic system.

Perfusion mechanism 268 may be modified to achieve similar perfusion orto change the exposure response time. For example, similar perfusion maybe obtained by disposing perfusion channels on opposing sides oftransverse channel 316, or disposing both perfusion channels on opposingwall 288. Alternatively, or in addition, the exposure time may beincreased or reduced by moving perfusion channel 310 closer to, orfarther from, main flow stream 314. Example 3 shows a perfusion channelthat empties directly into the focusing buffer stream.

FIG. 8 shows additional aspects of microfluidic system 250. Theseadditional aspects include macrofluidic reservoirs, and valves and/orpumps of the control layer that control fluid flow within themicrofluidic network.

Macrofluidic reservoirs allow system 250 to interface with themacroscopic world. Each reservoir or well functions as a fluidic inletor outlet connected directly to at least one microfluidic channel.Fluidic inlet-well A, shown at 330, provides for particle input,generally as a cell suspension. Fluidic inlet-well B, shown at 332,holds a focusing buffer, which is split into two focusing channels, 334,336, that ultimately form converging flow streams 272, 276. Fluidicoutlet-well C, shown at 338, holds output liquid, generally wasteliquid, that flows through the system. Well C accepts fluid from one orboth of fluid channels 340, 342. Fluidic inlet-wells D and E, shown at344 and 346, may hold first and second reagents for exposure to trappedcells. Fluidic inlet-well F, shown at 348, holds the shield buffer thatblocks exposure of the reagents until desired.

Control layer interfaces are numbered one through eleven. Each interfaceacts as a gas inlet to regulate opening and closing of one or morevalves. Interface seven controls cell input valve 350. Similarly,interface eight controls fluid channel 340, determining whether mainflow stream 314 bifurcates or is a single stream. Interfaces nine, ten,and eleven control valves 352, 354, 356, which regulate inflow ofreagent or shield buffer from fluidic inlets D, E, and F, respectively.Interfaces 1 through 3 and 4 through 6 control sets of values, shown at358 and 360, respectively. Valves within each set are actuated in adefined sequence to pump liquid by peristalsis from inlets B (valve set360) or D-F combined (valve set 358).

System Production

System 250 may be formed using any suitable method. In an exemplaryapproach, the system is formed by layering and fusing microfluidic layer252, control layer 254, and a substrate layer, formed, for example, by acover slip (not shown). Specifically, in this approach, the microfluidicand control layers are molded by soft lithography and then fused. Next,the resulting fused multilayer structure is bonded to the cover slipsubstrate layer. Finally, microfluidic channels are wetted withdeionized water.

System Operation

System 250 may be used to load, position, and/or retain particles, suchas cells, using any suitable method. In an exemplary approach, valves 7,9, 10, 11 are closed, and the remaining valves, including the pumpvalves, are opened. Wells B and F are loaded with focusing and shieldbuffers, respectively, wells D and E are loaded with reagents, and wellA is loaded with a cell suspension. Valve 7 is then opened, afterensuring that waste well C is at least partly empty, enabling cells toflow towards well C. At this point, no liquid flows from wells D, E, andF. Buffer flows from well B to well C, and cells flow from well A towell C. The cells flowing out of well A are focused in the center ofcombined flow stream 286 (see FIG. 7) by focusing fluid streams comingfrom well B, thereby flanking cells flowing from well A. The focusingfluid streams 272, 276 increase the likelihood that input cells willenter retention chamber 270, which is placed near where focusing occurs.The focused stream of cells is split into two streams adjacent theretention chamber. Each stream flows in a direction orthogonal to thefocused stream and opposite to each other, as described above. The trapis placed at a point of the flow stream below where the stream splits,so that the velocity of flow is lower than in the rest of the channel,therefore increasing the likelihood that retained cells are viable. Oncea sufficient number of cells are captured, valve 7 is closed to stop theflow of cells from well A.

System Protocols

System 250 may be used for any suitable protocols or proceduresinvolving positioned and/or retained particles. In a exemplary protocol,cells are exposed to reagents in wells D and/or E, as described below.This protocol is exemplified by successive exposure of retained cells tofirst and second reagents, such as a cell stain specific for dead/fixedcells and a cell fixative, respectively; see FIGS. 9-11.

The system is readied for perfusion as follows. First, valve 8 isclosed, so that the flow of focusing buffer from well B no longer issplit adjacent retention chamber 270. As a result, the focusing buffermoves predominantly or exclusively along main flow stream 314, which isunbranched (see FIG. 5). Next, pumps that control valve sets 354, 356are activated and run through the entire protocol. A suitable frequencyfor valve closure is about 60 Hz.

Shield buffer flow is initiated as follows. Initially, valves 7-11 arein a closed position, so that only focusing buffer from well B flowstowards waste well C. Then, valve 11 is opened, so that shield bufferflows from F to C and focusing buffer flows from B to C.

Flow of the first reagent, in this case Trypan blue, is initiated asfollows. Valve 9 is opened, so that fluid flows through both valves 9and 11. Valves 7, 8, and 10 are maintained in their closed positions.Since the shield buffer is flowing, the first reagent is spaced from thecell retention chamber by the shield buffer. Therefore, thisconfiguration readies the system for perfusion and may be used to washthe fluidic network without exposing the cells to either of the firstand second reagents.

Perfusion of the first reagent is initiated as follows. Once the fluidlines are washed with the first reagent, the shielding buffer is turnedoff, and the cells are exposed rapidly to the already flowing firstreagent. Specifically, valve 11 is closed, joining already-closed valves7, 8, and 10. In contrast, valve 9 remains open. In this way, the shieldbuffer no longer separates the flow stream of the first reagent and thecell retention chamber, allowing the first reagent to perfuse the cells.

After a suitable exposure time, the first reagent is washed out of thecell retention chamber as follows. Valve 11 is opened to restart flow ofthe shield buffer. In addition, valve 9 is closed to stop flow of thefirst reagent, joining already-closed valves 7, 8, and 10. In somecases, valve 9 may be left open to facilitate repeated exposure of thecells to the first reagent over a short time interval. FIG. 9 showsabout twenty Jurkat cells 380 in retention chamber 270 after exposure toa dye, Trypan blue, that stains fixed cells and a shield buffer to washaway the dye. Debris 382 is stained, but cells 380 are unstained.

Flow of the second reagent, in this case methanol, is initiated asfollows. Valve 10 is opened, joining already-open valve 11. Valves 7, 8,and 9 remain closed. This configuration is used to wash the fluidicnetwork with the second reagent without exposing the trapped cells tothis reagent.

Perfusion of the second reagent is initiated as follows. Valve 11 isclosed to turn off flow of the shielding buffer, joining already closedvalves 7, 8, and 9. Valve 10 remains open, to expose cells 380 to thesecond reagent, in this case methanol, thus fixing the cells. FIG. 10shows cells 380 being perfused with methanol. There is an opticallydetectable boundary 384 between the methanol 386 and the focusing buffer388, caused by their distinct indexes of refraction.

After a suitable exposure time, the second reagent is washed out of thecell retention chamber as follows. Valve 11 is opened to initiate flowof the shield buffer. In contrast, valve 10 is closed, to joinalready-closed valves 7, 8, and 9.

Cells 380 are then exposed for a second time to the first reagent,followed by washing with the shield buffer, as follows. The sequence ofvalve manipulations are as described above, except that valve 9 is leftopen during washing with shield buffer to show a shielded flow path ofthe first reagent. Now, since the cells have been fixed andpermeabilized by methanol, they stain with the dye carried in the firstreagent. FIG. 11 shows cells 380 stained blue after their secondexposure to Trypan blue and subsequent washing with shield buffer. Theshielded flow path 390 of the first reagent, Trypan blue, is visiblefocused between shield buffer 392 and focusing buffer 388.

The microfluidic system demonstrated here can be used for any suitableassay, such as screening compounds against a small population of cells,with the size of the small population be selected to be statisticallyrepresentative of cell behavior. The particles may include cells and/orbeads, among others. The cells may be nonadherent and/or adherent cells,either in suspension or attached to a substrate provided by themicrofluidic system. The beads similarly may be nonadherent or adherent,and may be used to carry samples, reagents, and/or cells, among others.

Embodiment 2

FIGS. 11A and 11B show a system 400 for measuring interaction betweenseparated, but proximate particles. Such interaction may be provided bydiffusible materials released by a first particle (or particlepopulation) and received by a second, separated particle (or particlepopulation). These diffusible materials may include cell-secretedhormones, viral particles, cell components released by cell lysis,and/or so on. The diffusible materials may produce changes in the secondparticle or particle population that are related to any measurableparticle or population characteristic, such as cell identity, geneexpression, apoptosis, hormone secretion, growth, and/or the like.Alternatively, or in addition, such communication may include long, thinprocesses extending from cells, such as axons and/or dendrites.Exemplary particle characteristics are described further in SectionsVIII and XII above.

System 400 may be formed by disposing two versions of system 250 in atail-to-tail configuration. Accordingly, each individual subsystem 250may include a retention mechanism 266, an individually controlledperfusion mechanism 268 for introducing reagents to each group ofcaptured particles, and an input flow stream 274 for carrying particlesand/or buffer to the retention mechanism. However, system 400 alsoincludes communication passages 402 that provide fluidic communicationbetween each retention mechanism 266 and retention chamber 270.

Communication passages 402 may be size-selective channels configured toprevent movement of retained particles, generally cells, between eachsubsystem 250. However, passages 402 are configured to allow movement orpassage of any smaller material released from the retained particles(such as molecules, polymers, molecular complexes, and/or smallerparticles, such as viruses), or of processes, such as axons and/ordendrites, extending to, from, and/or between retained cells.Furthermore, perfusion mechanisms 268 may be used to determine theeffect of reagents on cell-cell communication mediated by passages 402.

FIG. 11B shows an alternative embodiment of paired retention mechanisms266, mechanism 404, that may be included in system 400. Mechanism 404includes paired retention mechanisms 406, dimensioned to trap singleparticles 408. Retention mechanisms 406 are fluidically coupled throughcommunication passages 402. Accordingly, communication betweensingle-cells may be analyzed using mechanism 404.

Embodiment 3

FIG. 11C shows a retention mechanism 410 that may be used in system 250or any other suitable microfluidic system to form a positioned,two-dimensional array of retained particles. Mechanism 410 includes anarray of individual traps 412 oriented to receive particles from inletchannel 414. Traps 412 form a two-dimensional array of particleretention sites. Traps 412 may have any suitable configuration,including staggered rows, as shown here, orthogonally arranged rows andcolumns, or irregular configurations. (In some embodiments, some oftraps 412 may be positioned in alternative planes (e.g., in front ofand/or behind the plane of the drawing) to form three-dimensional arraysof retained particles.) Each trap 412 may be dimensioned to hold one orplural particles and may include size-selective channels or similarfeatures to allow fluid to flow through portions of the traps. Traps 412may be disposed within a common chamber 416 having an single or pluraloutlet channels 418 (such as chamber 270, described above, or chamber1970 of Example 10 below), within a chamber having no outlet besides aninlet channel, or within a channel, such as transverse channel 316,described above, among others.

Example 3 Microfluidic Systems for Parallel Retention and/or Treatmentof Particles

This example describes microfluidic mechanisms and systems that positiona plurality of particles and/or reagents at discrete transverse regionsand flow paths within a channel or flow stream; see FIGS. 12-13K. Thispositioning may allow parallel retention of distinct particles atadjacent, but distinct, sites and/or parallel exposure of particles atthese sites to distinct reagents.

Background

Biological analyses benefit from a capability to directly compare thephenotypes of two or more cells or groups of cells, under similar ordistinct treatment regimens. However, in the macroscopic world, suchcells or group of cells often are treated at distinct, relatively widelyspaced sites, such as different tissue culture dishes or wells of amicrotiter plate, potentially exposing the cells to undesireddifferences in treatment conditions. Accordingly, such analyses may needto be averaged over many experiments to achieve meaningful results.Therefore, it would be desirable to have a microfluidic system thatpositions, treats, and analyzes particles or groups of particlesadjacent one another at a microscopic level, to allow more consistentand efficient side-by-side comparisons.

Description

The microfluidic systems described in this example position a pluralityof particles or (particle populations) and/or reagents along distinct,transversely disposed flow paths or regions within a channel or flowstream. The transversely disposed flow paths may be defined byintroducing the particles and/or reagents into the channel alongdistinct laminar flow paths, by joining separate inlet channels (orinlet flow streams) carrying the particles and/or reagents. These flowpaths may abut one another or may be spaced apart by one or pluralspacer fluids, such as buffers. These spacer fluids may follow one orplural interposed flow paths formed by one or plural inlet channelsinterposed between the inlet channels that carry the particles and/orreagents.

The transversely disposed flow paths may be used to carry distinct (orsimilar) particles to distinct retention sites or chambers within thechannel. The distinct retention sites may retain distinct (or similar)particles for exposure to the same reagent. For example, the distinctparticles may be exposed to reagents, such as modulators and/or labels,to compare characteristics of the particles, such as response to themodulators, labeling characteristics, and/or so on. Thus, the positionof each retention site may be used to identify the correspondingparticle(s) retained at that position. For example, one retention sitemay be used to hold a control particle(s), as a reference, and anotherretention site may be used to hold a particle(s) of interest, allowingthe control particle(s) and the particle(s) of interest to be compareddirectly. Alternatively, one retention site may hold a bead(s) carryinga reagent, and another site may hold a cell(s) to be analyzed. In thisapproach, cell components released by cell lysis or secretion then maybe analyzed for interaction with the reagent held by the bead.

Alternatively, or in addition, transversely disposed flow paths may beused to expose similar (or distinct) particles to distinct reagents andto identify each reagent or exposed particle based on position.Particles may be retained at positionally distinct retention sites,either inputted from distinct reservoirs or a single reservoir. Next,the retained particles may be contacted with distinct reagents carriedto the distinct sites by transversely disposed flow paths. Thetransversely disposed flow paths may be formed by a set of inletchannels distinct from, and/or overlapping with, inlet channels thatintroduced the particles. Position of the retained particles identifieseach of the distinct reagents exposed to the particles. In someembodiments, the distinct reagents may include a compound with a knownactivity that acts as a reference, and one or more test compounds forcomparison.

The microfluidic systems of this example may allow more efficient andmeaningful use of microfluidic space for comparative analysis ofparticles and/or reagents.

In certain embodiments, a junction between two inlets and an outlet maybe used to transiently expose or perfuse particles, preferably cells,with selected reagents. By alternating the inlet flow between plus andminus reagent flows, the downstream conditions of the outlet will changein proportion to the rate of flow between both inlets.

Embodiment 1

FIGS. 12 and 13 show a microfluidic system 420 (Embodiment 1) forretaining separate populations of particles, and exposing thepopulations to one or more selected reagents.

Description of Embodiment 1

System 420 is formed by multilayer soft lithography, generally asdescribed above (for system 250) in Example 2 and below in Example 13.Here, particle positioning region 422 is shown as red rectangles,input/focusing channels 424 as blue regions, and perfusion channels 426as red lines. The dimensions of each region or channel and/or the numberof channels may be selected based on particle size, reagent deliveryvolume, and/or the number of separate populations to be retained, amongothers.

System 420 differs from system 250 of Example 2 in several aspects.First, system 420 includes more than one reservoir for holding andintroducing particles. Thus, inlets 1 and 2, shown at 428, 430,respectively, connect to particle input channels 432, 434. Second,system 420 includes three focusing channels 436, 438, 440, andcorresponding reservoirs or inlets for holding buffer (not shown). Thefocusing channels, also referred to as spacer channels, may be used toflank and separate the particle input channels. Third, system 420 hasmore than one retention chamber 442, with the chambers generallypositioned adjacent each other below confluence 444, where input flowstreams 446 join. Fourth, system 420 spaces retention chambers 442 fromwall 448, thus forming proximal and distal diverging flow streams 450and 452, respectively.

Applications of Embodiment 1

System 420 may be used as follows. Inlets 1 and 2 are loaded withdistinct suspensions of particles, such as different cell types, andinlets corresponding to focusing channels 436, 438, 440 are loaded withfocusing buffer. A pump(s) is started that drives flow of the focusingbuffer through the focusing channels. Valves that control the flow ofparticles from inlets 1 and 2 are opened. Particles enter confluence444, but are focused to spaced, intermediate, laminar flow streams 454,456, shown in FIG. 13, by flow from the focusing or spacer channels.Apertures 458, 460 of the retention chambers are aligned with particleflow streams 454, 456, respectively, to receive one or more particlesfrom the corresponding flow stream. By taking advantage of the laminarflow properties of fluids in system 420, the five streams flow togetherbut remain substantially distinct. Mixing of the fluids is limited todiffusion, which in the case of large particles, such as beads or cells,is very slow.

FIG. 13 shows the laminar flow pattern extending from confluence 444through divergence junction 462. Focusing flow streams 464, 466, 468flank and separate particle streams 454, 456, thus guiding particlescarried by these streams toward retention chambers 442. Flow streams injunction 462 may diverge above (464, 468), below (466), and/or within(470) retention chambers 442. Microchannels 472 within each retentionchamber pass fluid but retain particles.

After a sufficient number of particles have entered each retentionchamber 442, analysis of the particles may begin. Flow from inlets 1 and2 may be terminated, and flow may be converted from a divergent patternto a unitary flow path, by closing valve 474, as described above foroperation of system 250 in Example 2. Next, the trapped particles may beperfused with buffer/reagents from perfusion channels 426. In system420, perfusion channel 476 discharges fluid directly upstream of theretention chambers. This configuration may provide more rapid perfusionof trapped particles with reagents than system 250 of Example 2 above,because the outlet end of channel 476 is very close to the retentionchambers, feeding more directly into the unitary flow path produced bythe focusing buffers.

System 420 may be modified by changing various parameters. For example,the number of particle input-streams and/or focusing streams may bevaried, along with the number of retention chambers, to trap additionalparticle populations or individual particles. Thus, three or moreparticle input-streams may be used to trap three or more types ofparticles in three or more retention chambers. These three or moreretention chambers may be disposed in any suitable arrangement,including linear and staggered (e.g., triangular configurations). Insome embodiments, the size of the retention chambers may be varied, forexample, so that only one or a very small number of particles aretrapped in each chamber (see embodiment 2 of this example, and Examples4-7, 11, and 12 below). Furthermore, as described below, focusingstreams and spacer channels may be eliminated in some cases withoutsubstantial cross-contamination of particles between particle streamsand retention sites.

Embodiments 2 and 3

FIGS. 13A-C shows two alternative embodiments of system 420, systems 480(Embodiment 2) and 480′ (Embodiment 3), for retaining and treatingparticles at separate, but adjacent sites. Similar to system 420described above, system 480 or 480′ may be used to selectively input andretain one or plural particles at each of plural retention sitespositioned at discrete positions transverse to a flow direction within achannel. However, system 480 or 480′ also may be used to separatelycontact retained particles with distinct reagents at distinct retentionsites.

Description of Embodiments 2 and 3

System 480 includes an input mechanism 482, a focusing or transversepositioning mechanism 484, a retention mechanism 486, an outputmechanism 488, a plurality of individually controllable and distincttreatment mechanisms 490, 492, and a release mechanism 494; see FIGS.13A and 13B.

Input mechanism 482 includes particle input channels 496, 498 andfocusing or spacer channels 1762, 1764, 1766, similar to those describedabove for system 420. Particles, such as cells, may be inputted frominput reservoirs “Cell 1” and “Cell 2” along particle inlet channels496, 498, to positioning channel 1768. Input mechanism 482 also mayintroduce focusing or spacer fluid, preferably buffer, from bufferreservoirs 1770, 1772, 1774 (“Buffer 1,” “Buffer 2,” and “Buffer 3,”respectively) along spacer channels 1762, 1764, 1766, respectively, topositioning channel 1768.

Transverse positioning mechanism 484 may be determined by inletchannels. More specifically, the relative spatial configuration in whichthe inlet channels 496, 498, 1762-1766 join positioning channel 1768,along with relative sizes of, and/or flow rates from, these inletchannels, provides transverse positioning mechanism 482. Positioningmechanism 484 places each individual flow stream from each inlet channelin a laminar flow path based on this spatial configuration. Accordingly,particles from reservoirs Cell 1 and Cell 2 are spaced from each othercentrally in positioning channel 1768 by buffer from inlet channel 1764and laterally from each channel wall by buffer from inlet channels 1762,1766, as described above for system 420.

Retention mechanism 486 includes a plurality of single-particleretention sites, here referred to as “Trap A” and Trap B″ (see FIG.13B). Trap A and Trap B each are positioned to retain a particleintroduced by one of the two particle reservoirs, Cell 1 and Cell 2, andcarried at correspondingly distinct, transverse positions alongpositioning channel 1768; see FIG. 13B. Accordingly, Trap A ispositioned to retain a particle introduced from Cell 1, and Trap B fromCell 2. Particles not retained may be carried past retention mechanism486 to output mechanism 488, along central outlet (waste) channel 1776or flanking outlet (waste) channels 1778.

Treatment mechanisms 490, 492 provide exposure of retained particles todistinct reagents, indicated as Reagents 1-4; see FIG. 13B. A particleretained in Trap A may be exposed to Reagent 1 and/or 2 (controlled byvalves V2 and V3), and a particle retained in Trap B may be exposed toReagent 3 and/or 4 (controlled by valves V6 and V7). These reagents maybe stored and delivered (sequentially and/or simultaneously, in anydesired proportion and for any desired time) using any suitablemechanism, such as those described above in Example 2 and below inExample 8. Reagents from each treatment mechanism may be separatelyaddressed to a corresponding retention site, by transverse positioningof reagent flow streams entering positioning channel 1768. Reagents flowtoward central outlet channel 1776, but occupy a discrete portion of theentire flow stream within positioning channel 1768 and transversechannel 1780 due to laminar flow. Accordingly, reagents from treatmentmechanism 490 may be restricted to the left side of positioning channel1768 in FIG. 13B (and thus Trap A), whereas reagents from treatmentmechanism 492 may be restricted to the right half of the channel (andthus Trap B). Optionally, spacer buffer from central reservoir 1772,Buffer 2, may flow between reagents delivered by the treatmentmechanisms, reducing the probability of any reagent crossing over, andthus contaminating, the noncorresponding retention site.

Release mechanism 494 enables release of retained particles. Afterrelease, the released particles may be analyzed further and/orcollected, and/or the retention sites may accept a new set of particlesfor another round of treatment and analysis. Release mechanism 494, maybe operated by valve V4, to produce a localized reverse or dislodgingflow that propels the retained particles out of the retention sites.Release mechanism 494 is similar to the release mechanism describedbelow in Example 7. However, in contrast to the release mechanismdescribed below, retention sites in the present example are spaced fromreverse flow channels 1782.

FIG. 13C shows selected portions of a modified version of system 480,system 480′. System 480′ is distinct from system 480 in at least twoaspects. First, retention mechanism 1784 includes retention chambers1786, 1788 that are larger than the retention sites of system 480, andthus are capable of holding plural particles. Second, treatmentmechanisms 1790, 1792 include reagent inlet channels 1794, 1796 thatintroduce reagents into transverse channel 1798, rather than positioningchannel 1800. This altered position of the reagent inlet channels movesthe reagents farther from retained particles, but may facilitate washingout reagents toward outlet channels 1802 after exposure. However, duringtreatment, reagents from inlet channels 1794, 1796 are still positionedtransversely relative to the general direction of fluid flow towardcentral outlet channel 1804. Accordingly, reagent inlet channels maydeliver reagents at any suitable sites that allow laminar flow-basedlocalization of reagents.

Systems 480 and 480′ may be modified in any suitable aspect. Forexample, a single population of particles, such as from a single inputreservoir, may be retained at plural distinct retention sites, such asTrap A and Trap B, and then the sites separately exposed to distinctreagents introduced by distinct treatment mechanisms. Alternatively, orin addition, inlet channels provided by treatment mechanisms andparticle input mechanisms may overlap or converge upstream of a commonpositioning channel, such as positioning channel 1768 or 1800.

Applications of Embodiment 2

Exemplary operation of system 480 is described below using cells. System480 may be readied for operation by loading the input reservoirs withcells and buffers and equilibrating channels with the buffers, asdescribed in other examples.

Trap A and Trap B may be loaded as follows. Valves V1, V4, and V5 areopened, and valves V2, V3, V6, and V7 are closed. Five flow streamscoming from each of the five reservoirs meet before Trap A and Trap B inpositioning channel 1768. The cells from reservoirs Cell 1 and Cell 2are directed to their respective Traps A and B. Fluid and unretainedcells flow past retention sites along divergent flow paths toward aplurality of outlet channels 1776, 1778.

Once a cell (or cells) is retained in each retention site, valve V4 isleft open, and valves V1 and V5 are closed. Closing valve V1 blocksinput of additional cells, and stops flow from lateral buffer reservoirs1770, 1774. Closing valve V5 stops divergent flow, so that buffer (fromcentral buffer reservoir 1772 (Buffer 2)) flows to central outletchannel 1776 along a unitary path.

Distinct reagents may be delivered to the retained cells as follows.Valve V4 is left open, and all other valves remain closed. Both pumpsare running. Valve V2 and/or valve V3 may be opened to address Reagent 1and/or 2 to Trap A. Valve V6 and/or valve V7 may opened to addressReagent 3 and/or 4 to Trap B. Valves may be partially opened asdescribed in Example 8 to provide a desired mixture of reagents. Bufferfrom reservoir 1772 flows past Traps A and B to outlet channel 1776 andmay be used as a barrier between the streams of reagents addressed toTraps A and B. At any suitable time, valve V5 may be closed to releasethe retained cells.

Exemplary Results with Chips Produced According to Embodiment 2

System 480 was tested as described below. Microfluidic chips werefabricated according to system 480 of FIG. 13A and used for analysis offlow patterns and particle treatment efficacy with colored and/orfluorescent dyes.

FIGS. 13D-F show dye patterns formed by colored dyes introduced usingeach treatment mechanism and a Bowing spacer buffer to separatereagents. In each figure, Trap A holds a 10 μm bead, and Trap B two 6 μmbeads. FIG. 13D shows a dye pattern formed by green dye delivered fromeach treatment mechanism and an orange dye-labeled spacer bufferdelivered by reservoir 1772. The orange spacer buffer separate the twogreen dyes, and each green dye flows from its corresponding inletchannel 1806, 1808 to outlet channel 1776. Some green dye also travelsslowly along transverse channel 1780. FIG. 13E shows a dye patternformed by red dye delivered from treatment mechanism 490, green dye frommechanism 492, and orange dye from buffer reservoir 1772. FIG. 13F showsa dye pattern formed by red dye delivered from treatment mechanism 490,yellow dye from mechanism 492, and orange dye from buffer reservoir1772.

FIGS. 13G-13J show an analysis of treatment efficacy of single Jurkatcells captured in each of Traps A and B. FIG. 13G shows the two retainedcells 1810, 1812 prior to treatment. FIG. 13H shows exposure of eachcell to Trypan blue dye delivered by distinct treatment mechanisms. Thespacer buffer forms an uncolored column of fluid 1814 between the twoblue regions surrounding Traps A and B. Membranes of both cells areintact so neither stains efficiently with the dye. FIG. 13I showsexposure of cell 1810 in Trap A to methanol, to fix the cell, while cell1812 in Trap B is addressed with buffer. FIG. 13J shows the two cellsbeing exposed to the blue dye after fixation of cell 1810. Cell 1810 canno longer exclude the blue dye and is stained blue. Cell 1812 has notbeen in contact with methanol and is not stained.

FIG. 13K demonstrates that spacer buffers may not be required to preventcross contamination of particles and/or reagents during particle loadingand/or exposure to reagents. Each trap has been loaded with afluorescent bead 1816, 1818. Bead 1816 is addressed with a fluorescentdye, fluorescein, and bead 1818 with Trypan blue, using treatmentmechanisms 490, 492, respectively. No spacer buffer stream separates thetwo reagent streams, but the reagents do not substantially cross overand contaminate the other trap. It should be noted that the time fordiffusion of reagents (or particles) transverse to their laminar flowstreams is limited by the relatively short time that the laminar flowstreams are in contact before passing Traps A and B. Accordingly,analyses may be conducted with or without spacer streams, with spacerstreams being used to lower the probability of cross-contamination.

Embodiment 4

FIG. 13L shows a portion of microfluidic system 1820 that may be used toseparately address particles and/or reagents to sets of particle traps.System 1820 includes a plurality of serially arrayed sets 1822, 1824 ofparticle traps 1826. Each set 1822, 1824 is disposed to a discretetransverse position with a fluid flow stream, in this case defined by achannel 1828. Accordingly, laminar flow streams carrying particles(1830, 1832) or reagents (1834, 1836) may be segregated to discretetransverse regions of channel 1828, so that each set 1822, 1824 isindividually addressed. In alternative embodiments, traps 1826 aredisposed in a transverse channel, such as channel 1798 or a chamber,such as a cell chamber with size-selective channel around its perimeter.

Example 4 Microfluidic System for Multiplexed Analysis of Particles inan Array

This example describes a microfluidic system that loads particles in aserially distributed set of particle retention sites, and separatelyaddresses reagents to each of these sites in parallel; see FIGS. 14-16.

Background

Cell analyses often involve the use of arrays of cells or cellpopulations. These arrays may be formed in microtiter plates, so thatindividual wells within the array can be treated distinctly, forexample, with distinct test compounds. During or after treatment, themicroplate arrays are analyzed in multiplex to measure properties ofcells within each individual well. However, such arrays are difficult toform reproducibly with microtiter plates when single cells or a smallgroup of cells are placed in each well. Even if formed in microtiterplates, rapidly treating the cells in such microtiter plates, andmeasuring short-term consequences of such treatments, poses substantialtechnical hurdles. Therefore, a microfluidic system is needed that formsmore reproducible arrays of individual cells or small groups of cells atdistinct positions, and that allows separate, rapid treatment andanalysis of the cells at the distinct positions.

Description

This example describes a microfluidic system that serially traps smallsets of particles at preselected positions within the system, allowingtreatment of the trapped particles in parallel with desired reagents.Due to serial trapping of input particles, a single loading of particlesinto one inlet may be used to supply particles to an entire array oftraps. Thus, this design may be used to integrate a large number oftraps into a single system. This microfluidic system also reduces thenumber of control lines required, as single control lines regulate setsof fluidic channels, such as perfusion channels, that individuallyinterface with each of the traps. Accordingly, single control linesprovide parallel control for fluidic delivery to, or output from, eachof the traps. Such parallel control allows similar particles that areretained by each trap to be individually treated with distinct reagents.Furthermore, such parallel control allows all traps to be fluidicallyconnected during particle loading, but then fluidically isolated duringparticle treatment and measurement. This arrangement of the trapsenables the fabrication of larger microfluidic systems that may besuitable for use in high-throughput drug discovery. For example, system510 has a footprint of 2 by 4 cm. By increasing this density somewhatand increasing the number of traps over twenty-fold, at least 128 trapsmay be disposed on a single substrate of 8 by 12 cm, allowing each ofthe 128 traps to be addressed by two distinct reagents, with a total of256 reagents per substrate.

FIG. 14 shows a microfluidic system 510 for forming and analyzing anarray of particles. System 510 may be formed by any suitable technique,such as multilayer soft lithography, to include at least two distinctlayers: (1) a microfluidic network layer 512, shown in blue and orange,and (2) a control layer 514, shown in pink. Channels having distinctwidths and/or cross-sectional shapes may be formed within each layerusing molds fabricated, for example, as described in Example 17.

Microfluidic layer 512 includes two orthogonally directed networks.Particle loading network 516 is used to input and position particles, sothat the particles are retained at a linear array of particle traps 518.Particle treatment system 520 is an array of parallel, individualperfusion networks 522 that intersect loading network 516 at individualparticle traps 518.

Particle loading network 516 includes an inlet 524, an outlet 526, and aloading channel 528 extending there between. Inlet well 524, labeled C,is a reservoir that receives and holds a particle suspension to beintroduced into network 516. Outlet well 526, labeled W, is a wastereservoir that receives and holds fluid and unretained particles thathave traveled through network 516. Loading channel 528 carries particlesbetween inlet well 524 and outlet well 526 to each of a plurality ofparticle traps 518 disposed along channel 528. Fluid is activelytransported along network 516 by a three-valve pump 530, labeled “pump1,” which is positioned near the terminus of network 516 to pull fluidthrough the network. Positioning the pump after the traps delayspotential damage to fragile particles, for example, due to compressionunder closing valves, until particles have passed all particle traps518.

Each perfusion network 522 directs fluid between perfusion inlets 532,traps 518, and treatment outlets 534. Perfusion inlets 532 are of twomain types: buffer inlet-wells 536, labeled “B,” and reagent inlet-wells538, labeled “R_(xy).” The buffer inlet-wells hold a buffer or otherwashing or maintenance liquid, such as water or a solvent. Based ontheir positions within particle treatment system 520, the bufferinlet-wells are either a terminal inlet-well 540 or an intermediateinlet-well 542. Terminal inlet-wells 540 feed fluid to only one trap,whereas intermediate inlet-wells 542 are shared between two adjacenttraps. Based on whether they are intermediate or terminal inlet-wells,buffer inlet-wells feed a main stream and/or a shielding stream. Thecontrol and function of these two streams are described further below.The reagent inlet-wells hold one of two (or more) reagents (or reagentmixtures) that may be precisely exposed to an individual trap. Reagentinlet-wells are labeled “R_(xy),” with “x” referring to trap assignmentrelative to the array of traps 518, and “y” referring to one of the tworeagents that can be directed to a given trap. For example, reagentinlet-well R₁₂ feeds the first of the plurality of traps (closest inletC) with the second of two reagent choices for that trap. Fluid thatpasses each trap 518 may be directed to a corresponding treatmentoutlet-well 534 or waste well, labeled here as W1-W6. For example,reagents from reagent inlets R₄₁ and R₄₂ flow past and/or through trapnumber 4 and are collected in waste well W_(x), where x=4.

Control layer 514 regulates fluid flow from perfusion inlet-wells 532with a limited number of control lines that act on many fluid channels544 in parallel; see FIGS. 14 and 15. A three-valve pump 546, “pump 2,”acts simultaneously on all inlet channels 544 that extend from perfusioninlet-wells 532, to actively drive fluid from these inlet-wells to andpast traps 518, and on to waste outlet-wells 534. Opening or closingeach of four perfusion valves, V1-V4, determines whether fluid actuallyflows through each of the specific types of inlet channels 544 withinthe perfusion system. Valve V1 regulates control line 548, whichincludes a plurality of individual valves positioned over each of acorresponding plurality of focusing channels 550 included among inletchannels 544. Similarly, valve V2 regulates control line 552, whichincludes valves that control each of a plurality of first-reagentchannels 554, valve V3 regulates line 556, which controls each of acorresponding plurality of second-reagent channels 558, and valve V4regulates line 560, which controls each of a corresponding plurality ofshield channels 562. Thus, opening or closing each of valves V1-V4provides unified, parallel control over flow of individual inlets toeach of the plurality of traps.

FIG. 15 shows a portion of system 510, including traps 2, 3, and 4, toillustrate in more detail the design and rationale for the switchingvalves. Insulation valves 564 function in the control layer to mediateswitching between particle loading network 516 and particle treatmentsystem 520. Insulation valve V5 controls a set of valves that block flowalong loading channel 528 at a position downstream of particle inlet 524(inlet C) and of the traps. Thus, activation of valve V5 fluidicallyisolates each trap and converts system 510 from a particle-loadingconfiguration to a perfusion configuration. In contrast, insulationvalve V6 controls a set of valves blocking flow to each individualtreatment outlet 534, preventing diversion of particles to treatmentoutlets during particle loading, when valve V6 is closed. Therefore,valves V5 and V6 are primary determinants of parallel versus serial useof system 510.

FIGS. 15 and 16 show details of the loading mechanism. Loading channel528 forms a divided flow path 564 at each trap 518. Thus, particlestream 566 diverges directly upstream of each trap 518, at a T-junction568, following divided flow path 564, and then converging to formreunited particle stream 566. At each T-junction 568, a subset ofparticles do not follow divided flow path 564, but flow instead directlyinto trap 518. Accordingly, each trap is loaded using a divergent-flowmechanism, as described above in Example 2, but, in system 510, withoutthe use of focusing-buffer streams during particle loading to focusparticle flow within channel 528. In this example, trap 518 includes aretention chamber similar to retention chamber 270 of FIG. 5-8 inExample 2. However, any suitable traps may be used, such assingle-particle traps described below in Examples 4-7, 11, and 12.

The subsequent perfusion of trapped particles uses shielding andperfusion mechanisms analogous to those of Example 2. Buffer flow fromeach buffer inlet 536 flows along focusing channels 550, into loadingchannel 528, and past trap 518 in a unitary flow path 572, shown in FIG.16 as a dashed path, analogous to focusing buffer stream 314 of FIG. 5.Unitary flow path 572 may perform a variety of functions, such asbathing trapped particles during treatment, providing a retaining forceon trapped particles during perfusion, and focusing inflowing reagentsand shield buffer, in their laminar flow streams, toward the trappedparticles. Similarly, combined first and second reagent channel 554/558and shield channel 562 determine precise exposure to first and secondreagents, as described above in Example 2.

Applications

An exemplary use of system 510 to load particles and expose theparticles to different reagents is described below. System 510 is formedand readied for use as described elsewhere in this Detailed Description.

Loading particles into each of traps 518 may be conducted as follows.Valves 1-4 and 6 are closed, and valve 5 is open. Pump 1 is running, andpump 2 is not. The buffer inlet-wells B, shown at 536, are loaded withbuffer, each of inlet-wells R_(xy) is loaded with a reagent, andinlet-well C is loaded with a cell suspension. After making sure thatthe waste inlet-wells 526 are empty, pump 1 is allowed to pull theparticles to the traps.

Conversion from a loading to a perfusion configuration may be carriedout as follows. Once each of the traps has its desired occupancy and/oris full, pump 1 is stopped and valve V5 is closed. Each trap is nowisolated. Next, Valve V6 is opened to allow fluidic access to wasteoutlet-wells 534. Then, valve V1 is opened to permit flow of buffer fromeach inlet-well 536.

Trapped particles are perfused with each of the first and secondreagents as follows. Pump 2 is started, running at a frequency of about60 Hz. This pump is running throughout the following treatments. Pumpingaction of pump 2 drives buffer through focusing channels 550, alongunitary flow path 572 past each trap 518, toward waste outlet-wells 534.Prior to perfusion, valves V2, V3 and V4 are closed, so that only nofluid flows from along shield channel 562 or reagent channels 554, 558.Flow of the first reagent and the shield buffer is initiated by openingvalves V2 and V4, while valve V3 remains closed. This valveconfiguration is used to wash the fluidic network without exposing thetrapped particles to the first reagents, because the shield bufferdirects the first reagent stream to a spaced flow path separated fromthe trapped particles. Once the fluid lines are washed with each of thefirst reagents, valve V4 is closed to stop from of the shield buffer,allowing each of the first reagents to contact trapped particles. Aftera desired duration of exposure to each first reagent, valve V2 isclosed, allowing the shield buffer to wash away reagent one, and rapidlyterminating exposure. Trapped particles may be exposed to each secondreagent in parallel by following a comparable series of steps, butopening and then closing valve V3 instead of V2. In alternativeperfusion strategies, particles may be exposed to both the first andsecond reagents simultaneously, by opening both valves V2 and V3together. Furthermore, particles may be exposed to any desired ratio offirst and second reagents by partially closing valves V2 and/or V3, asdescribed below in Example 7.

Example 5 Microfluidic Device for Forming and Analyzing a Particle ArrayUsing a “Cell Comb”

This example describes a microfluidic device for forming and analyzingarrays of small number of particles, such as cells; see FIGS. 17-20.

Background

In many applications, it is necessary to form an array of cell-analysischambers, with each chamber containing the same number of cells. Thesechambers allow multiple experiments, such as drug screens, to beconducted in parallel, in a consistent and comparable fashion.Currently, standard analyses use wells of microtiter plates as cellchambers, distributing an equal volume of a cell suspension to each ofthe wells. The size of these chambers and thus the number of cellsanalyzed has been decreasing in response to efforts to reduce the use ofspace, reagents, and cells in these analyses. Unfortunately, resultsfrom these analyses become increasingly variable as the average numberof cells per well decreases. For example, with 96-well microtiterplates, there generally are about 3000 to 5000 cells at the bottom of awell; with 384-well plates, this number drops to about 1000 cells; and,as researchers push for smaller and smaller assay volumes, such as with1536-well plates, this number drops further to only about 250 cells.These small average numbers of cells may lead to variations in theactual number of cells among wells of as high as 20%. Such variationslead to huge errors in the detected reaction signals. Accordingly, witheven fewer cells per well, for example, with single cell assays or whencells of interest are in limited supply, microtiter plates do notprovide an adequate cell-analysis chamber unless cells are counted toplace an equal number per well. Even then, microtiter plates aredeficient for performing rapid experimental manipulations. For example,early responses to treatment with a drug are difficult to measure withmicroliter plates, because adding and mixing steps cannot be performedvery rapidly. Therefore, many cell-analyses would benefit from systemsfor efficiently loading, rapidly treating, and analyzing small numbersof cells.

Description

FIG. 17 shows a microfluidic device 610 for forming an array of singleparticles or small groups of particles. Device 610 includes an inputchannel 612, a waste channel 614, and an array of filter channels 616extending between the input and waste channels. Device 610 also includesa fixed-volume particle chamber 618 formed in each filter channel 616,and a set of valves for sample handling (see below). Device 610 may bereferred to as a “cell comb” because the path for cell (particle) flowtakes the shape of a comb, with chambers 614 representing the teeth ofthe comb.

The components of a cell comb each have a distinct function. Inputchannel 612 carries input particles, such as a particle 620, to eachfilter channel 616. A filter 622 is disposed within, or adjoining, eachfilter channel. Filter 622 allows fluid to pass into waste channel 614,but retains particles 620 in a portion of filter channel 616 thatcorresponds to chamber 618.

Filter 622 may take various forms, provided as a component(s) separatefrom the walls of filter channel 616 and/or integral to these walls. Forexample, filter 622 may be formed by a porous membrane that is specificfor each chamber 618 or that is shared by two or more or all chambers618. Alternatively, filter 622 may be formed by smaller, “leak” channelswithin filter channel 616, or by posts, obstacles, or protrusions thatextend into a portion of filter channel 616, or that are disposedadjoining or adjacent an end of the filter channel. The diameter of thesmaller channels, or the spacing of the posts/obstacles, determines thesize of particle retained in chamber 618. Thus, as long as the diametersof these smaller channels, or the maximum spacing between theseposts/obstacles, are sufficiently less than the diameter of a particleto be retained, the particle will be confined to chamber 618 while fluidwill pass readily into waste channel 614. In addition, the passage offluid through the filter provides a retaining force to reduce or preventbackflow of particles into input channel 612.

The capacity and retention ability of each chamber 618 is defined atleast in part by filter channel 616 and filter 622. The diameter andlength of filter channel 616, coupled with the position of filter 622relative to filter channel 616, define the capacity of chamber 618.Accordingly, chamber 618 may be dimensioned to receive a fixed number ofinput particles 620, such as a single particle. Such input particles mayhave a common size, such as cells from a homogeneous cell population, orthey may have a range of sizes, such as cells from blood. In someembodiments, the diameter of filter channel 616 allows size-selectiveretention of a single particle. For example, the diameter may be largeenough to receive certain particles in a heterogeneous particlepopulation, such as red blood cells, but small enough to exclude others,such as white blood cells. Filter 622 also acts size selectively, asdescribed above, so in combination with chamber 618, individual filterchannels 616 may be designed to retain a single cell within a definedsize range. Alternatively, individual filter channels may be designed toretain a group of two or more cells, with each cell having a minimumsize that is retained by filter 622.

Pressure differences within device 610 create positioning and retainingforces for particles 620. Flow between input channel 612 and wastechannel 614 creates a positive pressure difference between the inputchannel and the waste channel across filter channel 616. As a result,particles are carried into chambers 618 by fluid and fill each of thechambers very rapidly. After the particles have filled some or all ofchamber 618, a set of valves may be used to isolate each chamber 618(see below). In particular, the closure of such valves may transformeach cell chamber into an isolated reaction chamber, with a fixed numberof particles for analysis.

FIGS. 18-20 show valves, additional filters, and analysis sites that maybe used with, or added to, device 610 for manipulating the contents ofindividual chambers 618.

FIG. 18 shows a device 630 that is similar to device 610, but thatincludes a separate analysis site 632 opposing each chamber 618. A sitevalve 634 controls access to analysis site 632, and a pair of inputvalves 636 isolates each chamber 618 along input channel 612. The leftpanel of FIG. 18 shows a loading configuration for each of valves 634,636. Here, site valve 634 is closed (indicated by an “X”) to preventinput particles 620 from entering analysis site prematurely, and inputvalves 636 are open to allow particles to access each chamber 618. Theright panel of FIG. 18 shows repositioning of retained particle 620 toanalysis site 632. Here, site valve 634 is open, but input valves 636are closed. Particle 620 is displaced from chamber 618, by fluid flowingin reverse across filter channel 616 from waste channel 614, rather thaninput channel 612. Since input valves 636 are closed, fluid and particle620 flow orthogonally to input channel 612, into analysis site 632.After particle 620 is delivered to analysis site 632, site valve 634 isclosed to isolate the particle fluidically from other particles. Inother embodiments, additional fluidic lines may be used to deliverreagents to analysis site 632, or analysis site 632 may be a blindchannel that is preloaded with such reagents.

FIG. 19 shows a device 650 that is similar to device 630 of FIG. 18, butthat includes switchable filters 652. Switchable filters 652 may beswitched between a closed, filtering position, shown on the left, and anopen, nonfiltering position, shown on the right. After particle loading,switchable filters 652 are opened to direct particle 620 to an analysissite. Such a switchable-filter design allows unidirectional flow acrossfilter channel 616 to both retain and release particle 620. Accordingly,fluid flow from input channel carries out each both retention andrelease, using particle-laden fluid during retention, and particle-freefluid during release. Waste valves 654 are closed before switchablefilter 652 is opened to direct particle 620 to analysis site 656.Switchable/regulatable filters may be formed by size-selective channelsthat are formed on valve membranes. With this arrangement, deflection ofthe valve membranes may move the size-selective channels in or out offiltering position by pressure exerted through a control layer.Alternatively, or in addition, size-selective channels may be adjacentto, or flanking, valve membranes, as described below in Example 26.

FIG. 20 shows another device 660 with a switchable filter 652. In device660, waste channel 614 includes a series of waste filters 662 thatfunction in place of waste valves 654 in device 650. Waste filters 662play a dual role in allowing waste to flow down waste channel 664, whiledirecting particle 620 toward analysis site 666. The passages ofanalysis sites 666 may serve as waste channels.

Applications

Cell combs, described in this example, may be useful in a variety ofapplications. For example, cell combs may be useful in drug discovery,serving as replacements for microtiter plates in cell assays to providetighter control of the cell numbers. With current technology, thefabrication of each cell chamber in a cell comb device can be carriedout with precision. Therefore, cell assays may be performed with anarray of cells formed using this device, with reduced signal variationfrom chamber to chamber, even with single-cell assays. Cell combs may,more generally, be used with a variety of micron-sized particles, inaddition to cells, such as fluorescently or enzymatically coated beads.This device also can operate in gas phase, as long as the size of theparticles of interest is larger than the pore size of the filter units.Cell combs also can be cascaded so that objects of different sizes arefiltered out at different stages.

Example 6 Particle-Retention Mechanisms

This example describes mechanisms for retaining particles, usingparticle traps that are spaced from their corresponding substrates; seeFIGS. 21-23.

Background

One goal of microfluidic systems is the capability of retainingparticles at preselected positions for subsequent treatment andanalysis. Traps that perform such retention functions may performoptimally if they have minimal effects on fluid flow; otherwise, flowpatterns around the traps may be disrupted, slowing or reducing particleand reagent entry into the traps. Examples 1 and 2 above describe trapsthat may be used to retain single particles or groups of particles.However, these traps have limited flow through the traps themselves. Forexample, trap 180 of Example 1 includes blocks P and Q that reduce orprevent cross-flow on either side of a single retained particle.Similarly, retention chamber 270 of Example 2 includes relatively narrowmicrochannels 300 that may restrict fluid flow substantially. Thus,there is a need for an alternative trap that may be positioned closer toparticle input flow streams without disrupting flow patterns, whileallowing quicker and more efficient access by reagent and washing flowstreams.

Description

This example describes retention mechanisms having improved fluid flowproperties. These mechanisms are positioned downstream of a particleflow stream, near the point at which the particle flow-stream divergesat a T-junction. These mechanisms have been dimensioned to trap a singleparticle; however, they alternatively may be dimensioned to trap two ormore particles. The microfluidic system with respect to which eachretention mechanism is illustrated, particularly positioning mechanism264 and perfusion mechanism 268, is described above in Example 2. Thisearlier example describes suitable fluid flow paths, and the operationof the positioning and perfusion mechanisms. However, the retentionmechanisms presented in this example may be combined with any othersuitable microfluidic mechanisms for particle analysis.

Embodiment 1

FIG. 21 shows a microfluidic system 710 for positioning, retaining,and/or perfusing a single particle, in accordance with aspects of theinvention. Portions of system 710 that are molded from distinctphotoresist layers are shown as distinct colors, as described above (seeintroductory section of Examples). Retention mechanism 712 includes atrap 713, shown in turquoise, positioned centrally in T-junction 714, ina spaced relation from distal wall 716. Here, view 718, on the topright, is a schematic representation of trap 713, with points ofsectional view indicated; view 720, on the middle right, is a horizontalsectional view near the top of retention mechanism 712; and view 722, onthe bottom right, is a vertical sectional view nearer the side ofretention mechanism 712. Trap 713 extends downward from roof 724 as aU-shaped block 726. This block includes a recess 728 that acts as aretention site for a single particle. The block extends toward substrate730, in this case formed of glass, but remains in a spaced relation, inthis case about 5 μm apart from the substrate, to form a flow channel732 that extends under all of block 726. Thus block 726 forms astalactite-based trap with a potential flow stream below its entirebottom surface 734.

Embodiment 2

FIG. 22 shows another microfluidic system 740 for positioning,retaining, and/or perfusing a single particle, in accordance withaspects of the invention. View 742 shows a color-coded schematic of asystem 740, whereas view 744 shows a photograph of an actualmicrofluidic system formed according to view 742, but flippedhorizontally. System 740 includes a trap 746 positioned centrally atT-junction 714. Trap 746 is spaced from distal wall 716, disposing anyretained particle quite close to perfusion channel 748 for very rapidexposure to reagents (see Example 2 for a more complete description ofthe perfusion mechanism). Trap 746 includes a retention site 750 forholding a particle, flanked by trap channels 752, shown in turquoise,that extend to the edges of trap 746. Thus, fluid can enter retentionsite 750 and flow laterally out trap channels. View 754 shows thestructure of trap 746 schematically. Trap 746 includes three rectangularcolumns 756 that extend down to substrate 730, bridged by channelforming portion 758, shown in dotted outline in view 754, which extendsdown to 5 μm from substrate 730. Cross-sectional views 762, 764, 766show the structure of trap 746 in more detail.

Embodiment 3

FIG. 23 shows yet another microfluidic system 790 for positioning,retaining, and/or perfusing a single particle, in accordance withaspects of the invention. System 790 includes a particle retentionmechanism, trap 792, that abuts distal wall 716, in alignment withparticle stream 794 focused down input channel 796. Trap 792 includes aretention site 798, which is twenty μm in height, and flanked byretention blocks 800 that are spaced from substrate 730 by about 5 μm.View 802 shows a line representation of trap 792, but includes a portion804 of microfluidic system outside of distal wall 716. Sectional views806, 808 show how retention blocks 800 extend outward and downward fromdistal wall 716 and channel roof 810, but form a trap channel 812 thatextends under entire bottom surface 814 of the trap. Thus, trap 792 isstructured as a stalactite.

Views 816, 818 are two photographs taken of trap 792 at different depthsof focus. In view 816, the focal plane is near the substrate surface,showing sharp lines at corners 820, where the microfluidic layer 822contacts substrate 730. The bottom perimeter 824 of blocks 800 is blurrybecause bottom surface 814 is raised above substrate 730 (see also views806, 808). In view 818, the focal plane is slightly higher, raised about5 μm, placing bottom perimeter 824 in focus. Now, corners 818 are out offocus.

Example 7 Mechanisms for Reusable Microfluidic Systems

This example describes mechanisms that promote reuse of microfluidicsystems, including mechanisms for release, collection, and/orresuspension of particles; see FIGS. 24-28.

Background

Microfluidic systems often are designed for single use. Such single-usesystems may be used to retain and analyze a single cell or multiplecells, but they then are not or cannot be used again because the cell orcells interfere with analysis of newly introduced cells. Thus, thesesingle-use systems then are discarded, and additional single-use systemsmust be initialized for additional analysis. This approach is not anefficient use of the single-use systems. Moreover, this approach wastesmacroscopic volumes of cells and reagents, and is time consuming forinitialization. Thus, there is a need for a reusable microfluidic systemthat releases retained particles after their analysis, freeing thesystem (or cells) for additional analysis.

Description

This example describes microfluidic mechanisms that enable formation ofreusable microfluidic systems. These microfluidic mechanisms include (1)a particle release mechanism, (2) a particle collection mechanism, and(3) a particle suspension mechanism. The particle release mechanismremoves a particle(s) from a trap, generally after treatment and/oranalysis in the trap. The release mechanism may provide a force thatpropels particles out of the trap at any selected time. The particlecollection mechanism may be used to collect particles discharged by therelease mechanism. Collected particles may be cultured, measured,treated, and/or discarded. The particle suspension mechanism reducesparticle settling in an inlet well, so that a single loading ofparticles into the inlet well produces a relatively constant particleflow from the inlet well over time. These three mechanisms alone, or inany suitable combination, may enable more efficient and economical useof microfluidic systems for particle analysis.

Embodiment 1

FIG. 24 shows a microfluidic system 850 having a particle releasemechanism 852 and a particle collection mechanism 854, in accordancewith aspects of the invention. The general design of system 850 is asdescribed in Example 2, and elsewhere in this Detailed Description,including a particle focusing mechanism 856, a particle retentionmechanism or trap 858, and a perfusion mechanism 860. These particlefocusing and perfusion mechanisms are at least substantially equivalentto positioning and perfusion mechanisms 264, 268, respectively, shown inFIG. 5 of Example 2. System 850 may be formed as described elsewhere inthis Detailed Description. The meaning of each colored region of system850 also has been described above, and therefore will not be repeatedhere.

FIG. 25 shows trap 858 in more detail. Trap 858 may be dimensioned forcapturing a single particle and is similar to trap 746 of FIG. 22,described above, except that trap 858 disposes channel 862 againstdistal wall 864, in contrast to trap 746, which spaces channel 752 awayfrom distal wall 716.

Particle retention and treatment are essentially as described forExample 2 above, but the operation of a slightly different control layer866 is described here for clarity. Control layer 866 includes valvesV1-V4. Valve V1 corresponds to valve 8 of FIG. 8, described above, andis used to convert between divided and unified flow paths. Valve V2controls particle release mechanism 852; its function is describedbelow. Valves V3 and V4 control fluidic flow to waste reservoir 868 andparticle collection mechanism 854, respectively. During particle loadinginto trap 858, valves V1, V2, and V3 are open, and valve V4 is closed.During reagent delivery by perfusion mechanism 860, valves V1 and V4 areclosed, and valves V2 and V3 are open.

Particle release mechanism 852 may be used at any time to releaseparticles, particularly after use of perfusion mechanism 860 and/ormeasurement of trapped particles. Release mechanism 852 operates by adislodging flow to propel retained particles out their confinement intrap 858; see FIGS. 24 and 25. The dislodging flow originates in areservoir channel 870 that is fluidically connected to trap 858 using asize-selective channel 872. Size-selective channel 872 has a diameterthat prevents entry of particles but that does not restrict passage offluid to, or from, reservoir channel 870.

Fluid flow through size-selective channel 872, and thus particlerelease, is controlled by valve V2 (see FIG. 24). Valve V2 is acontrol-layer valve disposed over reservoir channel 870. When valve V2is closed, reservoir channel is compressed, forcing fluid outwardthrough size-selective channel 872 into trap 858. This releases trappedparticles, propelling them out of trap 858 into a flow stream, such asmain flow stream 874, shown in FIG. 25, which carries the particles awayfrom trap 858. Typically, in use, the focusing buffer pump is running,the reagent valves are closed, and the shield buffer is running. Thus,the main flow stream goes from the buffer wells to the cell culturearea, described below. When valve V2 is opened, reservoir channel 870expands, bringing fluid in through size-selective channel 868 andrefilling the reservoir channel.

Embodiment 2

FIG. 26 shows a system 880 for retaining and releasing groups ofparticles, in accordance with aspects of the invention. System 880generally is similar to system 850 (compare with FIG. 25), but withseveral exceptions. First, trap 882 includes a much larger retentionsite 884 than trap 858, capable of holding a group of particles. Thus,walls 886 extend substantially into cross channel 888, and each wallincludes three size-selective channels 890, rather than the one presentin trap 858. Moreover, trap 882 is wider than trap 858, so multipleexpulsion channels 892 are used to release particles from confinement intrap 882, rather than one. Second, perfusion channel 894 has been movedslightly away from focusing channel 896 to ensure effective delivery ofreagents to, all particles in trap 882.

Released particles generally may be discarded or saved for furthertreatment and/or analysis, for any trap size or configuration. Particlesto be discarded may be carried toward waste reservoir 868 by openingvalve V3 and closing valves V1 and V4 (see FIG. 24). Alternatively,particles to be saved may be carried toward particle collectionmechanism 854 by opening valve V4 and closing valves V3 and V1 duringparticle release. Thus, valves V3 and V4 provide a sorting mechanism 898to selectively discard or collect each individual particle or group ofparticles.

Once a retained particle has been released, system 850 may be readied totrap another particle. Toward this end, valve V4 is closed, if it-wasopened during particle release, and valves V1, V2, and V3 are opened.System 850 then is ready to receive another particle.

Embodiment 3

FIGS. 24 and 27 show a particle collection mechanism 854, in accordancewith aspects of the invention. Collection mechanism 854 includes aninlet channel 904, a retention area 906, filter channels 908, and anoutlet 910. Inlet channel 904 carries released particles towardretention area 906 when valve V4 is open during release. Fluid flowsthough retention area 906 to outlet 910 by passing through filterchannels 908, which act as size-selective channels that prevent releasedparticles from flowing to the outlet. Thus, released particle arecollected in retention area 906. When the collected particles are cells,the retention area may be used to culture cells to promote cell growth,differentiation, and/or response to a treatment, such as by perfusionmechanism 860. Alternatively, the retention area may be operativelyconnected to a measurement system for particle analysis, and/or may be asite of particle lysis or further treatment. In some embodiments, inletchannel 904 may be connected to other channels (not shown) that allowreagents to be introduced to retention area 906 separate from particleretention, treatment, and analysis at trap 858. Alternatively, or inaddition, reagents may be introduced by perfusion mechanism 860 and/orfocusing channel 896. Particles collected in retention area 906 may bereleased by reversed flow to send them up inlet channel 904 or byforming collection mechanism 854 so that a valve (or valves) replacessome of the filter channels.

Embodiment 4

Standard particle input mechanisms, such as inlet-well 330 of FIG. 8,are sufficient for single-use microfluidic systems. However, thesemechanisms may be inadequate for reusable systems. In reusable systems,it may be desirable to load a suspension of particles into aninlet-reservoir(s) at the beginning of an analysis, and then to use thatsame suspension as a source for multiple particle loadings and analyses.Unfortunately, during such extended analyses, particles typically settleout of the suspension, so that the particle input concentrationdecreases with time, increasing the amount of time required to loadparticles. Thus, there is a need for a mechanism for maintainingparticles in suspension in an inlet reservoir during extended analyses,to allow repeated loading and analysis of particles from thissuspension.

FIG. 28 shows a particle suspension mechanism 920 that may be integratedinto reusable microfluidic systems, such as systems 850 and 880described above. This suspension mechanism helps to maintain particlesin suspension and/or helps to resuspend settled particles during thecourse of analyses with a reusable microfluidic system. Mechanism 920includes an inlet reservoir 922, recirculation channels 924, and pumpingvalves 926. Inlet reservoir 922 receives and stores particle suspensionsduring analyses. Thus, reservoir 922 may be an interface with themacroscopic world. Recirculation channels 924 are joined at each end 928to the base of reservoir, but are spaced from the reservoir at anintermediate portion 930. Pumping valves 926 are regulated by thecontrol layer, and are coordinated to peristaltically pump fluid throughrecirculation channels 924, as described elsewhere in this DetailedDescription. Accordingly, fluid in reservoir 922 flows away from, andthen back to, reservoir 922, continuously acting to mix the contents ofreservoir 922 and thus to maintain the particles in suspension.Therefore, a more stable concentration of particle flows from outlet 932over time.

Example 8 Microfluidic Mechanisms for Adjustable Reagent Delivery

This example describes mechanisms for adjustably diluting reagents sothat reagents may be delivered to particles at a range of reagentconcentrations, for example, as a gradient; see FIGS. 29-30.

Background

Studies of cells frequently involve dose-response analyses to determinehow the cells respond to a range of concentrations of a reagent, such asa drug. These dose-response analyses may be used to determine a varietyof qualitative and/or quantitative information, including an effectivedose, a half-maximal response dose, a lethal dose, a dose to produce amore specific response, and so on. In many analyses, a reagent ofinterest is prepared as a high concentration stock solution, and thenvarious volumes of the reagent are dispensed to provide a range ofdoses. However, this approach may not be suitable with microfluidicsystems, because it may not be practical to dispense metered volumes ina microfluidic system and because it may require a mixer to mix and thusdilute such a dispensed volume. Thus, there is a need for a microfluidicmechanism that dispenses a premixed reagent at a range of selectedconcentrations, using a small number of reagent stocks.

Description

This section describes two exemplary dilution mechanisms, havingindependent (Embodiment 1) and coordinated (Embodiment 2) control.

Embodiment 1

FIG. 29 shows an adjustable dilution mechanism 960 for combining firstand second reagents at a range of concentrations, in accordance withaspects of the invention. Dilution mechanism 960 includes a microfluidiclayer 962 having first and second reagent reservoirs 964, 966, and firstand second controllable flow channels 968, 970 acting as outlets for thereservoirs. The controllable flow channels narrow and meet at a junction972 to form a common mixing channel 974. Reagents are mixed in mixing ordiffusion channel 974, generally by diffusion of reagents into theadjacent flow stream(s). Thus, mixing channel 974 may be substantiallynarrower than flow channels 968, 970, generally about 1 to 20 μm. Incontrast, flow channels 968, 970 are wide enough to be controlled byvalves, with an arcuate cross-section. Here, fluid flow from eachreservoir is independently controlled by control layer 976, viathree-valve pumps 978, and shutoff valves 980; however, fluid flow inother embodiments may be controlled by other control mechanisms.

Dilution mechanism 960 is used to combine first and second reagents, R1and R2, in a desired ratio based on the rate at which each pump movesfluid through flow channels 968, 970. Thus, reagent R1 may beintroduced, for example, at 100%, 50%, 20%, 10% and 0% of reservoir 964concentration, by running pumps 976 and 978 at relative pumping flowrates of 1:0, 1:1, 1:4, 1:9, and 0:1, respectively. Valves 980 may beused to override the pump and/or to modulate the effect of a specificpump rate, as described below. To improve control, the adjustabledilution mechanism may use relatively precise control of pump speed anda large number of control lines in the control layer.

Embodiment 2

FIG. 30 shows another adjustable dilution mechanism 990 for combiningfirst and second reagents at a range of concentrations, in accordancewith aspects of the invention. Dilution mechanism 990 is structuredsimilarly to dilution mechanism 960, as indicated by components withidentical numbering. However, dilution mechanism 990 uses a single pump978, generally at a constant pumping rate, to coordinately drive flow ofboth reagents. Furthermore, mechanism 990 uses adjustable valves 994,996, rather than shutoff valves. Closure of adjustable valves iscontrollable by regulating the pressure used to deflect the adjustablevalves. Thus, each adjustable valve may be independently adjusted with asuitable pressure to provide a desired partial obstruction to flowchannels 968, 970, and thus a desired flow rate and reagent mixture indiffusion channel 974. A simple dilution of a first reagent may becarried out by using an appropriate solvent or buffer as the secondreagent.

Applications

The dilution mechanisms described above may be used as part(s) of anysuitable microfluidic device, for any suitable applications. Forexample, dilution mechanism 990 may be used in microfluidic system 250in FIG. 8 of Example 2 to prepare and deliver a desired mixture ofreagents for particle perfusion, by providing empirically determinedpressures to valves 9 and 10.

Example 9 Microfluidic Sorting Mechanisms Based on Centrifugal Forces

This example describes mechanisms for sorting particles based on theirmass, density, and/or other properties; see FIGS. 31-38.

Background

Microfluidic analyses of particles may benefit from or even requiresorting crude or heterogeneous input populations of particles into theircomponents. For example, the input population may be a mixture of singlecells, cell clusters, and/or cell debris. Alternatively, or in addition,the input population may be a mixed population of distinct cell types.In these cases, sorting may separate single cells from clusters anddebris, and cells of one type from cells of another, type. Opticalsystems may be used to actively sort individual particles according totheir different optical properties, such as fluorescence intensity.However, these optical systems require that the input particles beconstantly monitored and actively directed to distinct sorting binsbased on optical properties. Thus, there is a need for a microfluidicsorting mechanism that separates distinct particles, potentiallypassively, based on different physical properties of the distinctparticles.

Description

This example describes mechanisms for passively sorting particles basedon physical differences between the particles, such as mass, density,shape, and/or surface characteristics, among others. These mechanismsare passive, exploiting the centrifugal forces exerted on flowingparticles during a sharp change of direction, rather than activemonitoring and switching. These mechanisms are described anddemonstrated as part of simplified fluidic systems lacking valves andother functional mechanisms. Instead, fluids are moved through thesesystems by pressure differences produced by liquid columns havingdifferent heights in input and output reservoirs. However, these sortingmechanisms may be integrated into any suitable microfluidic system.

Embodiment 1

FIGS. 31 and 32 show a microfluidic system 1020 having a sortingmechanism 1022 that separates particles according to physicaldifferences between the particles, in accordance with aspects of theinvention. Here, mechanism 1022 sorts particles from inlet reservoir1024 into one of three outlet or sorting channels 1026. These sortingchannels lead to distinct outlet reservoirs 1028, labeled here asoutlets 1-3. The sorting channels in this embodiment have a minimumwidth of about 50 μm and a height of about 17-18 μm. However, moregenerally, mechanism 1022 may be formed with any suitable dimensions.Furthermore, mechanism 1022 may sort particles from any suitable source,such as a microfluidic treatment or analysis, into any desired number ofoutlet channels and/or other microfluidic mechanisms or structures, suchas culture chambers, retention mechanisms, perfusion mechanisms, and/orthe like.

Mechanism 1022 includes structures that act sequentially along a flowstream. First, hydrodynamic focusing region 1030 acts to focus particlesfrom particle inlet channel 1032 into a narrow stream. Two sidereservoirs 1034, 1036, each filled with a focusing fluid, such as abuffer, are connected to inlet channel 1032 using focusing channels1038, 1040. Focusing channels 1038, 1040 may have different widths, andthus different flow rates, to asymmetrically position the narrow streamin the inlet channel. Second, acceleration region 1042 narrows the widthof the channel to increase the flow velocity and further focus particlesinto a single stream. Third, curved region 1044 bends sharply to givethe input particles an angular velocity and a radial acceleration.Fourth, a separation region 1046 is positioned after curved region 1044.Separation region 1046 widens into a larger chamber with a number ofreceiving or sorting channels 1026 that act as sorting bins to segregatesorted particles. In separation region 1046, particles are distributedbased on their mass (weight). The tendency of particles to continuemoving in a straight line increases with mass, so that heavier particlesmove to the outside of the flow stream, and lighter particles remaincloser to the center of the flow stream. Accordingly, in thisembodiment, the heaviest particles tend to distribute more to receivingchannel 1048, the lightest particles to receiving channel 1050, and theintermediate-mass particles to receiving channel 1052. In some cases,other physical properties of the particles, such as density, shape,and/or surface properties, among others, also may contribute to therelative distributions of particles between these receiving channels.

The sorting capabilities of sorting mechanism 1022 may be modified byaltering one or more of several potential sorting parameters. Thesesorting parameters may include the extent of narrowing of theacceleration region, the radius of curvature of the curved region, theangle of broadening of the separation region, and/or the number ofreceiving channels/bins, among others. These parameters may impart suchcapabilities as improved resolution, separation into a different numberof sorting channels (bins) and/or resolution of a different range ofparticle weights, densities, etc.; among others.

Embodiment 2

FIG. 33 shows a microfluidic system 1060 having a sorting mechanism 1062with modified sorting parameters, in accordance with aspects of theinvention. Sorting mechanism 1062 has a narrower acceleration region1064 than acceleration region 1042 of sorting mechanism 1022,potentially imparting greater velocity to the particles, and thus betterfocusing. In addition, sorting mechanism 1062 has a curved region 1066with a distinct radius of curvature relative to curved region 1044 ofsorting mechanism 1022. Furthermore, sorting mechanism 1062 has aseparation region 1068 having a greater angle of separation (subtendedangle) than separation region 1046 of sorting mechanism 1022, connectedto four, rather than three, sorting channels 1070.

Embodiment 3

FIGS. 34 and 35 show another microfluidic system 1080 having a sortingmechanism 1082 with modified sorting parameters, in accordance withaspects of the invention. Sorting mechanism 1082 has a narroweracceleration region 1084 than either region 1042 or region 1064,providing even greater velocity and focusing. In addition, sortingmechanism 1082 has a curved region 1086 with a smaller radius ofcurvature than curved regions 1044 and 1066 of FIGS. 31-33. Furthermore,sorting mechanism 1082 has a separation region 1088 with an even greaterangle of separation, compared to regions 1046 and 1068.

Applications

FIGS. 36-38 show experimental results demonstrating the ability ofsystems 1020 and 1060 to sort a mixed population of particles. In theseexperiments, the mixed population of particles was formed, prior toloading into an input reservoir, using two sizes (and types) ofparticles: beads with an average diameter of about 1 μm, and Jurkatcells with an average diameter of about 10 μm. These two sizes ofparticles are distinguishably labeled with distinct fluorescent dyes:the beads emit green light, and the cells emit red light.

FIG. 36 shows an image of particles being sorted using a sortingmechanism as described in this example. The particles are split into twostreams 1100 in the separation region. The lower stream is enriched forcells (red), and the upper stream is enriched for beads (green). Flow ofparticles through the system is powered by a 1-cm high column of fluidin the inlet reservoir.

FIGS. 37 and 38 show graphs of data obtained with systems 1080 and 1020,respectively, as each sorted the mixed population of beads and cells,described above. These graphs were generated by counting the relativenumbers of particles that entered each of two receiving channels. Thegraphs each plot the fraction of cells (blue diamonds) and beads (pinksquares) that distribute to the lower receiving channel, either sortingchannel 1102 or 1048, respectively. The ratio of cells to beads in thelower receiving channel is plotted in yellow. In both system 1080 and1020, a greater fraction of cells than beads are entering the lowerreceiving channel. In system 1080, about twice as many cells as beadsentered the lower receiving channel. In system 1020, this ratio wasslightly lower and more variable.

Summary

The systems shown in this example have the ability to passively enrichparticles based on sorting mechanisms that distinguish physicalproperties of particles. The approximately two-fold enrichment obtainedusing these systems may be sufficient to facilitate or improve somemicrofluidic analyses. Furthermore, each of these systems may bemodified and refined, and/or connected in series to improve enrichmentof desired particles.

Example 10 Microfluidic Systems for Manipulating Sets of Particles

This example describes microfluidic systems having relatively largechambers, in which larger sets of particles, such as adherent and/ornonadherent cells, can be retained, stored, cultured, treated, and/orreleased; see FIGS. 39-50D.

Background

The introduction and/or removal of particles into and out ofmicrofluidic systems, at macroscopic/microscopic interfaces, mayinefficient and/or harmful. For introduction, particles must be placedin suspension and often are introduced through an inlet reservoir.During this loading process, a substantial fraction of the particles maybe lost, which may be problematic if the particles are expensive and/orin limited supply, such as with cells from a clinical or forensicsample. Furthermore, during introduction and/or removal, particles maybe contaminated, for example, by exposure to contaminatingmicroorganisms, and/or damaged, for example, by evaporation of inlet- oroutlet-reservoir liquid. Accordingly, it is desirable to avoidrepeatedly introducing and removing particles from microfluidic systemsduring a sequential set of assays. Therefore, there is a need forchambers for storing, treating, maintaining, measuring, and/or inparticular, amplifying (i.e., culturing) particles, such as cells,particularly for serial analyses of particle populations. With suchchambers, these serial analyses could be conducted without transferringthe populations to a macroscopic environment between analyses.

However, such chambers need to address a number of problems or issuesrelated to their use with cells. First, these chambers may need aceiling height that does not interfere with cell movement within thechambers. In particular, the ceiling of larger chambers, particularlythose formed of elastomeric materials, may tend to sag, obstructing cellmovement. Second, these chambers may need a substrate that promotesadhesion, survival, and growth of adherent cells, when such cells arebeing used. Many adherent cells do not behave normally unless they areattached to a substrate. Third these chambers may need to pass mediaand/or reagents over cells in the chambers, without loss of, or damageto, the cells. Pumps that circulate fluid may crush fragile eukaryoticcells, and some filters that restrict cell movement may be clogged bycells and/or allow cells to pass. Fourth, these chambers may require anability for gas to diffuse into cell chambers, to maintain a proper pHduring cell growth.

Description

This example describes various microfluidic systems that address andsolve some or all of the problems and issues cited above. Thesemicrofluidic systems may be formed using multilayer soft lithography, asdescribed elsewhere in this Detailed Description and in theCross-References. Channels or chambers for particle storage, treatment,analysis, and cell growth are formed using molds fabricated as describedgenerally in Example 13, using plural layers of photoresist, whenneeded. Such molds may be used to construct channels large enough forcell entry and growth, for example, about 200 μm wide by about 20-35 μmhigh. Furthermore, as described below, such molds may be used to formparticle chambers of various dimensions. These channels and/or chambersmay be integrated into microfluidic systems that include valves, pumps,rotary mixers, filters; sorters, multiplexers, perfusion mechanisms,and/or additional particle retention sites, among others, to perform anysuitable analysis of particles.

Embodiment 1

FIGS. 39-43 illustrate exemplary microfluidic networks 1130 that includerelatively large chambers 1132 for retaining particles, in accordancewith aspects of the invention. These networks have been fabricated usingmultilayer soft lithography, with large chambers that did not collapse.These chambers have a height of about 36 microns. The chambers wereformed by a modified process using molds in which two layers, each ofabout 18 microns, were sequentially layered on top of a substrate, andselectively retained at the positions where the cell chambers wereformed. The chambers were rounded. This process produces a generallyarcuate (arch-like) cross-sectional configuration that may enhancestability. As a result, this process allows formation of chambers withwidth-to-height ratios less than about 10:1 that do not collapse. Incontrast, microfluidic channels having width-to-height ratios greaterthan 10:1 formed by a standard soft lithography process may collapsemore frequently.

The large chambers may be connected to an input reservoir 1134 and anoutput reservoir 1136. The input reservoir may connect to an inletchannel 1138 that bifurcates, as shown at 1140, to direct flow into eachof two channels 1142. Outlet channels 1144 extend from each pair ofchambers to join and carry fluid to output reservoir 1136. For moreefficient use of space and input reservoirs, some systems, such assystem 1146, share a common inlet reservoir 1148 for two pairs ofchambers. Thus, particles may be loaded into inlet reservoir 1148 todistribute the particles to each of four chambers. In other embodiments,an input reservoir may be fluidically connected to one, two, three,four, or more chambers using any suitable number of channels. Thechannels may extend directly between a particle reservoir and a cellchamber, or they may branch any desired number of times at any desirednumber of positions. The movement of fluid through these chambers may becontrolled by any suitable mechanism, such as valves and/or pumps, amongothers. For example, FIG. 44 shows a system 1150, in which an array ofnetworks 1130 are controlled in parallel by control lines 1152, 1154that regulate valves 1156 flanking each chamber 1132. In this case, eachof the eight valves shown is opened or closed in parallel throughactuation at control port 1158, either providing an open chamber forparticle loading, or a closed chamber for particle isolation,respectively.

Chambers 1132 may have any desired shape and size. Suitablecross-sectional shapes may include diamonds 1160 (FIGS. 39 and 41),rectangles 1162 (FIGS. 39, 42, and 43), squares 1164 (FIG. 39), circles1166 (FIGS. 39 and 40), ellipses or elongated circles 1168 (FIGS. 39,40, and 41), and/or the like. Suitable sizes are about 100 microns toabout 1 centimeter in diameter, depending on particle type, assay, andso on. Specific chambers shown in FIGS. 39-43 that have been constructedsuccessfully have diameters of from about 0.9 mm to 2.6 mm.

Chambers may be completely isolated from the substrate in theirinteriors, or they may be supported by columns, posts, or otherstructures. These columns or posts may project downward from the roof ofthe channel to contact the substrate, generally being integrally formedin the microfluidic layer during fabrication of this layer.Alternatively, or in addition, these columns or posts may project upwardfrom the substrate, being formed as a portion of the substrate or anaddition to the substrate. To be effective, the columns or posts shouldbe spaced adequately to avoid obstructing cell movement through thechambers, although more tightly spaced structures could be used to forma cell pen or other subchamber.

Embodiment 2

FIG. 45 shows a microfluidic system 1180 having a microfluidic network1130 through which fluid flow is more flexibly controlled. Specifically,fluid flow through chamber 1132 is controllable by two nested sets offlanking control valves 1182, 1184 that sit to both sides of chamber1132. A parallel pumping circuit 1186 is disposed as an parallel fluidpath 1188, having pump 1190 and extending from upstream and downstreamcell chamber 1132, at an intermediate nested-position between nestedvalve sets 1182, 1184.

System 1180 may be operated as follows. During cell (particle) loading,nested valve sets 1182, 1184 are opened and fluid flows passively frominput reservoir 1134 to output reservoir 1136, bringing cells to chamber1132. When a desired number of cells have entered chamber 1132, one orboth of valve sets 1182, 1184 are closed to isolate chamber 1132. Ifonly valve set 1182 is closed, pump 1190 may be activated to circulatefluid through a loop that include chamber 1132 and alternate fluid path1188, to prevent cell adhesion to the substrate, or to maintain a fluidflow over cells that have adhered. Alternatively, only valve set 1184may be closed, allowing fluid to flow between input and outputreservoirs using alternate, parallel fluid path 1188, to the exclusionof a path through chamber 1132. Thus, fluid channels may be flushed andre-equilibrated with any desired reagent. Once the fluid channels havebeen re-equilibrated, the desired reagent, valve set 1182 may be closedand the desired valve set 1184 may be opened, to actively pump thedesired reagent in a closed loop that includes chamber 1132. Forexample, the reagent may be a mixture of trypsin and EDTA, or anothersuitable detaching reagent. Pumping the mixture of trypsin and EDTAthrough the closed loop detaches adhered cells. Opening valve set 1182then allows the detached cells to be flushed from the system, either tooutput reservoir 1136 or to any additional microfluidic mechanism or setof mechanisms, as described throughout this detailed description.

Embodiment 3

FIG. 46 shows a microfluidic system 1210 with a cell chamber 1212 formedas a looped channel or ring structure, in accordance with aspects of theinvention. Cells (or particles) are introduced into chamber 1212 andretained there, either by balancing fluid height between input andoutput reservoir 1214, 1216, respectively, or by closing one or morevalves 1218 that interconnect these reservoirs. Partial closure ofvalves 1218, particularly valves adjacent or within chamber 1212, may beused to permit fluid flow, while preventing cell flow, past the valves.Once cells are loaded into chamber 1212, four valves 1220 may beactuated in an appropriate order to move fluid around chamber 1212

Embodiment 4

FIGS. 47-49 shows another microfluidic system 1240 with a chamber 1242formed as a looped channel or ring structure, in accordance with aspectsof the invention. System 1240 offers distinct networks for particleinflow/outflow—particle network 1244—and for reagentinflow/outflow—reagent network 1246. These distinct networks overlap atchamber 1242.

Particle network 1244 is used to load particles into chamber 1242 and toreceive particles flowing from chamber 1242. Particles are loadedinitially into input reservoir 1248, which feeds the particles intoinput channel 1250. Input channel 1250 flows into chamber 1242 Chamber1242 bifurcates and rejoins at outlet channel 1252. Outlet channel 1252carries fluid to output reservoir 1254. Fluid flow between reservoirs1248 and 1254 can be terminated at any selected time by closing one orboth of valves 1256 and 1258. Closing both valves fluidically isolateschamber 1242 from the remainder of particle network 1244.

Reagent network 1246 is used to move fluid, particularly fluid carryingreagents, through chamber 1242, while selectively retaining particles.Reagent network 1246 directs fluid and reagents from one or more reagentreservoirs 1260 through inlet channel 1262 into chamber 1242. Flow fromeach reagent reservoir 1260 is independently regulated by valves 1264,which control flow of a single reagent or a mixture of reagents. Desiredratios and/or dilutions of reagents may be formed by preciselycontrolling flow rate through each valve, for example, as describedabove in Example 8. Reagents entering chamber 1242 from inlet channel1262 follow a bifurcated path that rejoins at outlet channel 1266.Outlet channel 1266 carries fluid to waste reservoir 1268. Inflow oroutflow can be regulated with valves 1270, 1272, respectively, which maybe closed to isolate chamber 1242 from reagent network 1246,particularly during particle loading and/or removal. Furthermore, areagent pump 1274 may be used to pull reagents from reagent reservoirs1260 to waste reservoir 1268.

Reagent network 1246 blocks exit (and entry) of particles from (and to)chamber 1242, based on particle size. To achieve this, reagent network1246 interfaces with chamber 1242 using filtering mechanisms 1276. FIGS.48 and 49 show photographs of size-selective channels 1278 disposed inoutlet channel 1266, adjacent chamber 1242.

Chamber 1242 includes a chamber pump 1280 (see FIG. 47). Chamber pump1280 is used to circulate fluid through chamber 1242, for example, (1)to suspend cells (such as during detachment of adhered cells withtrypsin), (2) to move cells away from filtering mechanism 1276, reducingor preventing clogging of the mechanism, (3) to promote mixing withinchamber 1242, and/or the like.

An exemplary method for feeding cells in chamber 1242 is a follows. Oneof reagent reservoirs 1260 is loaded with about 20 μL media, and wastereservoir 1268 is loaded with about 10 μL media (or buffer). Thesereservoirs have the same diameter, so this asymmetrical loading givesreagent reservoir 1260 a fluid head of about 10 μL. Flow to equalizefluid heights subsequently transfers about 5 μL of media through chamber1242 to waste reservoir 1268 over the course of about 30 min. Particlenetwork 1244 may be used instead, or in addition, if the cells inchamber 1242 are adherent.

System 1240 allows extended culture of adherent cells. FIG. 50 shows NIH3T3 cells 1290 that are alive and adherent in chamber 1242, 3 weeksafter they were seeded. The field of cells shown has been tested forviability (top panel) and visualized for general morphology by brightfield illumination (bottom panel). A substantial majority of cells wasdetermined to be alive, as evidenced by lack of ethidium homodimerstaining (Molecular Probes; Live/Dead Viability Assay Kit), and to havenormal morphology. During the 3-week incubation, cells 1290 weresubjected to the passive-flow feeding regimen described above, repeatedonce every 2 days.

Embodiment 5

FIG. 50A shows a system 1910 for depositing cells (or other particles)in a microfluidic chamber 1912, based on an asymmetrically disposed flowpath. Particles and fluid flow into chamber 1912 from inlet channel1914. The particles and fluid may follow plural distinct flow paths1916, 1918 toward outlet channels 1920, 1922, respectively. One or morevalves 1924 may be used to select one or both of the flow paths.

Selection of asymmetrically disposed flow path 1916 allows a subset ofinputted cells to be deposited in chamber 1912. Main flow path 1916 maybe both asymmetrically disposed and nonlinear. Such a flow path definesa highest velocity main stream corresponding to main flow path 1916.However, some of the fluid also follows lower-velocity auxiliary streams(weaker flow streams) disposed more distally in chamber 1912, inquasi-stagnant region 1926. Accordingly, the subset of cells thatfollows the auxiliary streams within chamber 1912 tend to be depositedin chamber 1912 by settling out and contacting a substrate defined bythe chamber. Such contact diminishes the ability of fluid flow to movethe settled cells and may promote additional interactions between thesettled cells and the substrate, such as formation of a secretedextracellular matrix. In other embodiments, the subset of cells that aredeposited may be determined by varying any suitable parameters includingdegree of nonlinearity of flow path 1916, location of flow path 1916relative to the chamber, chamber dimensions, fluid flow rate, and/or thelike.

Embodiment 6

FIG. 50B shows a system 1930 that is based on system 1910 but includesadditional mechanisms and features. System 1930 includes an inputmechanism 1932, an output mechanism 1934, and a treatment mechanism1936. Input mechanism 1932 includes an input reservoir 1938 forintroducing cells and/or fluid, such as buffer or media. Outputmechanism 1934 includes an output reservoir 1940 that may receive fluidfrom outlet channels 1942 and/or 1944, provided by flow paths 1918and/or 1916, respectively. Valve 1924 may be operated to block flowalong path 1918, whereas valve 1948 may be operated to block flow tooutput reservoir 1940 from either flow path. Treatment mechanism 1936may include plural reagent reservoirs 1950, valves 1952 that regulateflow from each reagent reservoir, and a valve 1954 to regulatecommunication between entire treatment mechanism 1936 and chamber 1912.

System 1930 may be used to deposit cells as follows. Cells are inputtedby input mechanism 1932, generally with valve 1948 opened, and valve1924 closed. Cells travel along flow path 1916, with a subset followingauxiliary flow streams to be deposited in quasi-stagnant region 1926, asdescribed above.

Once a sufficient number of cells have been deposited within chamber1912, the deposited cells may be manipulated further as follows. Valve1956 may be closed and the contents of input reservoir 1938 replacedwith media to achieve a fluid head that is approximately equal to thatof output reservoir 1940, to produce no net flow between reservoirs (a“balanced flow” condition), and then valve 1956 may be reopened. Thedeposited cells may be incubated a suitable time period, such asovernight, during which time they may adhere by interaction with asubstrate defined by the chamber. Such adhered cells are retained withinchamber 1926. Alternatively, nonadherent cells may be used withoutattachment to chamber 1912.

Adhered (or nonadhered) cells may be treated with reagents from reagentreservoirs 1950 by operating treatment mechanism 1936. First, reagentsmay be introduced into chamber 1912 by opening one or more valves 1952,and valve 1954, to direct selected reagents along flow path 1958, alonga reverse of flow path 1916, and/or along outlet channel 1944. Next,chamber 1912 may be placed within a closed loop by closing valves 1948,1954, and 1956. Pump 1960 may be started to circulate reagent around theclosed loop, providing a mixing action that continuously perfuses cellsin chamber 1912 with reagent.

Embodiment 7

FIG. 50C shows a cell chamber 1970 that may be used to deposit (andretain) cells in one or two compartments 1972, 1974. Compartments 1972,1974 may be connected by radially arrayed, size-selective channels 1976to form a “spoked wheel” structure. Cells (or other particles) may beinputted from first input channel 1978 and deposited in compartment1972. Fluid may flow through size-selective channels 1976 to secondinput channel 1980. Alternatively, or in addition, additional cells,such as a distinct cell type, may be inputted from second input channel1980 to be deposited in outer compartment 1974, with fluid flowingtoward first input channel 1978. With each of the two compartmentsoccupied by distinct cell populations, cell-cell communication may beanalyzed by passage of released cell components (or extended cellstructures) through the size-selective channels between the twocompartments. In alternative embodiments, the first and secondcompartments may have any suitable geometry, such as interdigitatedfingers or intermeshed spirals, among others, to increase the area ofcommunication between the two compartments. Furthermore, additionalcompartments may be added to measure interactions between additionalcell types.

Embodiment 8

Cell chamber 1990 is a modified version of chamber 1970 that includes anoverflow capability. Here, inner compartment 1972 acts as a chamber thatis connected to overflow compartment 1992 by transverse passages 1994,in addition to size-selective channels 1976. Accordingly, input channel1978 may be used to direct most of inputted cells (or other particles)into inner compartment 1972 using entrance 1996. However, once innercompartment 1972 becomes filled, additional cells may travel alongtransverse passages, through overflow compartment 1992 and out outletchannel 1998.

Applications

The microfluidic systems described here may be used for the manipulationof adherent and nonadherent cells. For example, after, introduction to achamber, NIH 3T3 cells adhere to the substrate to retain the cellseffectively within the chamber. Once adhered, these cells remainattached to the substrate as fluidic flows are directed over thempassively and/or actively. These cells remain viable at a range of flowrates and valve closure pressures. However, cell viability may becompromised when higher valve actuation pressures are used, becausehigher pressures lead to complete valve closure. A valve that closesupon a cell can crush it. In particular, at high pumping frequencies,all cells within a population inside a ring may be crushed, since theyhave a high probability of being crushed. In this case, the ring maybecome filled with cell debris, which may be a starting point for assayson cell components. The nuclear membrane may or may not be compromisedby this treatment.

In general, manipulation of adherent cells on the chips is achieved inthe following manner. Adherent cells are prepared from seed flasks byreleasing the cells from the flasks, for example, by trypsinization,followed by washing, centrifugation, and resuspension in a standardtissue culture medium, such as DMEM or RPMI. Once a desiredconcentration has been achieved, cells are loaded using a manualpipettor into the input well and cells flow into the microfluidicchannel structures under the head flow generated by the column ofliquid. Once adhered, adherent cells can be resuspended in themicrofluidic channel by addition of trypsin-EDTA or other cell-detachingagents.

The microfluidic layer and substrate may be treated (or left untreated)to promote cell flow, cell viability, cell adhesion or nonadhesion, cellgrowth, and/or the like. Fluidic channels and/or the substrate may betreated with a nonionic detergent, such as TWEEN; a serum protein, suchas a serum albumin (e.g., BSA); whole or fractionated serum from anysuitable animal; extracellular matrix extracts, components, or mixtures,such as collagen, polylysine, SIGMACOTE, MATRIGEL, etc.; and/or thelike.

Example 11 Systems for Electrophysiological Analysis of Cells in aMicrofluidic Environment

This example describes microfluidic systems for positioning, retaining,treating, and/or measuring cells, particularly for electrophysiologicalanalyses; see FIGS. 51-58.

Background

Cell-surface membranes are an essential part of all cells, definingtheir extent, and separating and maintaining the differences between thecell interior (cytoplasm) and the extracellular milieu. Accordingly,controlling membrane permeability and the selectivity of ion movementacross membranes, mediated by ion channels and transporters, isfundamental to cell survival, cell physiology, and signal transductionmechanisms, particularly neurotransduction. Thus, many cell-surfacereceptors couple to ion channels and transporters, making measurement ofmembrane currents a very rapid and sensitive indicator of cellphysiology and receptor activity. Therefore, many drug assays benefitfrom or, in some cases, require a measurement of the effects of drugs onion currents, referred to as electrophysiology.

The preferred method for conducting electrophysiological analyses ofcells membranes is the “patch-clamp” analysis of individual cells.Typically, in this approach, a glass electrode with a diameter of about0.1-1 μm is electrically sealed against the membrane of a single cell,surrounding a membrane “patch” on the cell. The patch then may be leftintact, separated from the cell, “perforated” with channel-formingagents, or penetrated, based on the type of information desired. Withboth intact patches and patches separated from a cell, the size of thepatch and the density of channels in the membrane determine the numberof channels being analyzed. Thus, different sizes of patches may allow“single-channel recordings” from small regions of membrane, orrecordings from many of channels in “macropatch recording.”Alternatively, membrane patches can be perforated or penetrated tomeasure electrical properties of the entire cell membrane, in“whole-cell” patch-clamp studies. Perforated patches introduce achannel-forming agent, such as the antibiotics nystatin or amphotericinB, into the membrane. Perforated patches enable whole cell recording ofchannel activity with loss of larger cytoplasmic components. Penetratedpatches place an electrode inside a cell, so that the electrode and thecell's cytoplasm are continuous. Accordingly, penetrated patches alsoenable whole-cell patch-clamp recording.

Despite the importance of electrophysiology as an assay tool and thevariety of patch-clamp methods available for measuring electricalactivity at membranes, these methods require substantial time and skillfor their proper execution. In particular, each of these methodsgenerally is carried out manually, by a highly-skilledelectrophysiologist. The electrophysiologist must precisely position anelectrode against the membrane of each cell, and manipulate theelectrode and/or cell additionally to form a gigaseal and/or penetratethe cell. Accordingly, the electrophysiologist must devote considerabletime and energy to the execution of patch-clamp methods, making themexpensive and ill-suited to screening applications in which many samplesmust be studied. Thus, there is a need for a more automated system thatsimplifies cell manipulation and at least partially automates patchformation.

Description

This example describes microfluidic devices that allow measurements ofion channel activity. These devices position a single cell in abutmentwith an aperture, so that the cell's membrane forms a high resistance,gigaohm seal, termed a gigaseal, around the aperture. The gigasealallows channel currents across the cell membrane to be measured, by“whole cell” patch-clamp recording. Measurement of currents in thepresence and absence of potential modulators of channel activity, suchas agonists and antagonists of receptors that couple with channels,provides a rapid and sensitive method for testing these modulators.Since changes in channel currents often are transient, the device alsofacilitates rapid perfusion of the cell with potential modulators andwash solutions. This allows rapid exposure and removal of themodulators. The device may be configured as a system that simultaneouslyand/or sequentially analyzes more than one single cell (see, amongothers, Example 12).

Embodiment 1

FIG. 51 shows a microfluidic device 1310 for measuring ion currents, inaccordance with aspects of the invention. Device 1310 includes a planarpatch clamp electrode consisting generally of three layers: a substratelayer 1312, a fluidic layer 1314, and a base layer 1316.

Substrate layer 1312 includes one or more patchable orifices 1318, ofabout 0.1-5 μm, or about 1-5 μm in diameter. The perimeter of eachorifice forms a gigaseal with the membrane of a single cell beinganalyzed. Accordingly, substrate layer 1312 may be fabricated from anynonconducting material capable of forming a highly resistant seal, andmay be relatively hard. Suitable materials for the substrate layerinclude glass, silicon, and/or plastic, among others.

The substrate layer separates fluidic layer 1314 and base layer 1316.The fluidic and base layers each are filled with one or more buffersolutions that mimic the external and internal ionic environments,respectively, of single cells being analyzed. These buffer solutions maybe referred to as external and internal buffers, respectively. Themovement of ions through the cell membrane, effectively between thefluidic and base layers, creates currents that can be measured usingsensitive amplification equipment. The fluidic layer may be formed byany suitable technique, such as multilayer soft lithography, forexample, as described elsewhere in this Detailed Description. Thefluidic layer may be controlled by any suitable control mechanism, suchas an overlying microfluidic control layer 1320. The base layer may beformed out of any suitable material, such as glass, plastic, and/or anelastomeric material, among others. The base layer may be cut (punched),molded, etched, and/or embossed, among others, to (1) form a tight sealwith substrate layer 1312, and (2) form a reservoir holding internalbuffer that is in fluidic contact with each orifice and that accepts anelectrode and/or electrode plate, typically connected to suitablestimulation and recording equipment. In preferred embodiments, the boreof the patch clamp channel may be large enough to permit dislocation ordislodging of the particle from the patch clamp when fluid flow isreversed through the bore of the patch clamp channel.

Embodiment 2

FIGS. 52-58 shows a microfluidic system 1340 for single-cell patch-clamprecordings, in accordance with aspects of the invention. System 1340includes a fluid-layer network 1342 and a fluid control layer 1344, bothformed by multilayer soft lithography, for example, as describedelsewhere in this Detailed Description. Network 1342 and control layer1344 position a single cell over a patchable orifice or aperture formedby a substrate layer (see below). Positioning the single cellestablishes an appropriate buffer gradient between fluid-layer network1342 and a base-layer fluidic chamber, as described above for FIG. 51.Once a high-resistance seal is formed between the positioned cell andthe substrate, around the orifice, system 1340 allows the positionedcell to be perfused with one or more of a set of reagents, such asdrugs, ligands (for the case of ligand-gated channels), buffers withdistinct ionic compositions, and/or wash solutions. Perfusion of thesereagents permits rapid measurement of the effect of these reagents onthe electrical activity of the cell.

To carry out these functions, system 1340 includes several mechanismsthat cooperate serially and/or in parallel. A cell manipulationmechanism 1346 inputs, positions, and retains single cells. A cellperfusion mechanism 1348 exposes and washes the retained single cells ina precisely controlled manner using a set of reagent-input networks. Anelectrical monitoring mechanism 1350 electrically contacts both thefluid-layer network 1342 and a base-layer fluidic chamber (not shown) tomeasure current, voltage, and/or resistance of retained single cellsbefore, during, and/or after exposure to desired reagents and/orelectrical manipulations.

Cell manipulation mechanism 1346 itself includes a set of mechanisms,including a cell input mechanism 1352, a cell positioning mechanism1354, and a cell retention mechanism 1356. These mechanisms act in acoordinated fashion to manipulate single cells for patch-clampexperiments.

Cell input mechanism 1352 generally comprises any mechanism that actsthrough an input reservoir 1358 to introduce cells into fluid-layernetwork 1342. Input mechanism 1352 is similar to input mechanism 263 ofExample 2. Other suitable input mechanisms are described above, inSection IV.

Cell positioning mechanism 1354 generally comprises any mechanism thatacts to position single cells within microfluidic network 1342. Inaddition to simple flow channels, the cell-positioning mechanism mayinclude a focusing mechanism 1360. Focusing mechanism 1360 places inputcells in an input stream 1362 at a central portion of inlet channel1364, labeled “E1,” flanked by focusing flow streams from focusingreservoirs 1366, 1368, labeled “F1” and “F2.” Mechanism 1360 directsfluid from input and focusing reservoirs 1358, 1366, 1368 to junction1370 from three orthogonal directions. FIG. 53 shows an alternativecell-focusing mechanism 1372, in which cell-input and focusing streamsjoin at acute angles, forming an “arrowhead” configuration. Focusingmechanisms 1360 and 1372 are similar to aspects of positioning mechanism263 of Example 2.

Cell positioning mechanism 1354 stochastically segregates single cellsusing a divided-flow mechanism 1374, downstream from focusing mechanism1360 or 1372; see FIG. 54. Specifically, focused cells are directed downinlet channel E1 and encounter a divided flow path 1376. Divided flowpath 1376 directs fluid to a waste reservoir 1378 (see FIGS. 52 and 53)through outlet channels 1380, 1382 (labeled “W1” and “W2,” respectively,in FIG. 54). These outlet channels include a narrowed portion 1384 and asize-restrictive channel 1386 that determine the relative flow ratethrough each corresponding outlet channel. Narrowed portion 1384 has asubstantially larger diameter than size-selective channel 1386, so thatmost of the flowing fluid (and cells) passes through narrowed portion1384. However, some fluid passes through size-restrictive channel 1386,eventually bringing a single cell 1388 to the mouth of the channel.

Cell retention mechanism 1356 generally comprises any mechanism forretaining a cell at a desired position, generally adjacent an orificeand/or electrode(s). Here, the cell retention mechanism functions at thechannel mouth; see FIGS. 54 and 57. In particular, cell 1388 cannotenter size-restrictive channel 1386 because the cell is too large.However, the pressure drop across size-restrictive channel 1386 pullscell 1388 against the channel mouth, holding cell 1388 in position.Positioned cell 1388 may restrict or block flow through size-restrictivechannel 1386, so that additional cells no longer are urged towardchannel 1386. Cell 1388 also is positioned over an orifice 1390 (seeFIG. 56) defined by the substrate layer. In alternative embodiments,single cells may be positioned and retained over an orifice by anysuitable positioning and/or retention mechanisms, for example, thosedescribed elsewhere in this Detailed Description.

With cell 1388 in position over orifice 1390, flow from input reservoir1358 is terminated, but flow from focusing reservoir F1 and/or F2continues. Continued flow from F1 and/or F2 may be used to preventadditional cells from stopping near cell 1388, which might interferewith measurements. In addition, continued flow from F1 and/or F2 ensuresthat buffer in the region surrounding cell 1388 is refreshed. To performwhole-cell recordings, reservoirs F1 and/or F2, and generally inputreservoir 1358, are filled with external buffer, so that all of fluidicnetwork 1342 is equilibrated with external buffer. In contrast,base-layer chamber, below orifice 1390, is filled with internal bufferfrom a lower face (or side) of the base layer, generally prior to cellinput. The contents of these reservoirs could be reversed, if the cellis positioned on the opposite side of the aperture, or for reasons ofexperimental design.

Positioned cell 1388 is pulled against orifice 1390 by applying a vacuumfrom the base-layer chamber. This establishes a highly resistant seal,the formation of which can be measured as an increase in resistancebetween fluid-layer network 1342 and the base-layer chamber (beloworifice 1390) using electrodes in each chamber. Generally, fluid-layernetwork 1342 serves as a ground, and a recording electrode is positionedin the base-layer chamber. Once the seal is formed, the resultingpatched cell can be measured for its baseline electrical activity orproperties.

After establishing this baseline, and/or using an average or calculatedbaseline, the effect of reagents, such as drugs, may be tested usingperfusion mechanism 1348. FIG. 52 shows the general layout of mechanism1348, which includes a shield or wash reservoir 1394, and a series ofreagent reservoirs 1396, in this case five reservoirs, labeled D1-D5.Flow through inlet channels 1398 extending from reservoirs 1394, 1396 isactively promoted by a pump 1400 in control layer 1344. Pump 1400 actsin concert on all inlet channels 1398 to provide a uniform force fordelivering the reagents and wash buffer. In contrast, flow through eachindividual inlet channel 1398 is regulated by a corresponding controlvalve 1402 that determines whether fluid flows through the inlet channel1398. Valves 1402 are shown in more detail in FIGS. 53, 54, 56-58, wherethese valves are labeled V_(W), and V1-V5, corresponding to control ofwash reservoir (“W”) and reagent reservoirs D1-D5, respectively.

FIG. 55 show perfusion mechanism 1348 in more detail. Perfusionmechanism 1348 controls exposure of cell 1388 to each selected reagentusing a regulatable fluid sheath or shield, similar to that describedfor perfusion mechanism 268 of Example 2. Wash reservoir W is filledwith external buffer, and the buffer is flowed past cell 1388 from washinlet-channel 1404 by opening valve V_(W). Specifically, focusing bufferfrom F1 and/or F2 entering chamber E1 pushes the wash buffer in alaminar flow pattern or sheath flow 1406 over cell 1388, against wall1408. Because wash inlet-channel 1404 is closer to cell 1388 than any ofthe reagent inlet channels 1398, sheath flow 1406 spaces and preventscontact of reagents flowed from any of the reagent inlet channels. Uponclosing valve V_(W), any flowing reagent rapidly contacts the cell, andrecordings can be made as desired. Accordingly, cell 1388 may be exposedrapidly to any reagents in reservoirs D1-D5 in a controlled manner byselective opening and closing valves V_(W) and V1-V5, allowingmeasurement of electrical responses in a correspondingly rapid timeframe. Therefore, ligands introduced through reservoirs D1-D5 may beused to study their antagonist or agonist activity on ligand gatedchannels, among others.

Microfluidic system 1340 may be configured in many suitable ways. Forexample, reagent inlet channels may unite, entering chamber E1 through acommon port, as shown in system 250 of Example 2 (see FIG. 8). In thisway, each reagent is equally spaced by sheath flow 1406 of the washbuffer and thus will reach cell 1388 at the same time when the sheathflow is terminated. Furthermore, such a design would allow reagentmixing and dilution, as described above in Example 8. Alternatively, orin addition, a pump may be included to drive flow from input reservoir1358 and focusing reservoirs 1366, 1368. Furthermore, system 1340 may bemodified to be reusable by including a cell removal mechanism, asdescribed in Example 7. System 1340 may be modified additionally oralternatively to include a parallel or serial array ofretention/analysis sites, for example, as described above in Examples3-5, or below in Example 12.

Example 12 Microfluidic System for Multiplexed Analysis of Cells byPatch Clamp

This example describes microfluidic systems for performingelectrophysiological analysis on one or more cells out of a set ofsingle cells; see FIGS. 59-61.

Background

Patch clamping is an electrophysiological method that relies on theformation of a seal between a biological membrane (for instance, a cell)and an aperture. This seal may facilitate the measurement of smallcurrents created by the passage of ions across the membrane. However,the seal generally should be tight, since current leakage around theseal may interfere with, or prevent, measurement of the small currentsacross the membrane.

The efficiency of seal formation is an important issue for thedevelopment of automated, high-throughput devices for screening drugsbased on electrophysiological effects on cells. In manual patch-clampsystems, the efficiency with which cells can be successfully analyzedvaries, but very skilled technicians typically achieve properly sealedpatches at an efficiency of only about 50%. A similar efficiencyachieved by an automated device would require the device to“cherry-pick” wells containing properly sealed patches for use in drugscreens, limiting the utility of such a device. Furthermore, even whenproperly sealed patches are formed, more than one cell may need to beanalyzed to identify a typical or average cell response. Thus, there isa need for an automated device that more efficiently forms sealedpatches on cells, facilitating averaged analysis of multiple cells andreducing problems associated with cell-to-cell variation inelectrophysiological response.

Description

This example provides a multiplexed version of a single-aperturemicrofluidic device, with a defined number (“n”) of individuallycontrollable apertures. Each individually controllable aperture may beused to analyze a single cell by patch-clamp methods. Because only onepatched cell is required to form an effective seal for each experiment,the use of multiple apertures increases the probability of forming thisseal with the device. In addition, the device allows each aperture, andits associated cell, to be included in, or excluded from, an analysis.Thus, signals may be obtained from each individual cell that issuccessfully sealed by electrically isolating each correspondingaperture. Alternatively, or in addition, an “averaged” signal may beobtained from two or more of the individually controllable apertures,either by averaging separate measurements or measuring from two or moreapertures concurrently. Averaged signals may improve the robustness ofany data obtained.

Single-Aperture Embodiment

FIG. 59 shows a one-aperture device 1430 to illustrate how each of the napertures is structured. Device 1430 directs a single cell 1432 intoabutment with an aperture 1434. Aperture 1434 connects chambers 1436,1438. These internal and external chambers, 1436 and 1438, respectively,carry buffers whose compositions resemble that of the internal(cytoplasm) and external (extracellular) environments, respectively, ofcell 1432. A vacuum may be applied to internal chamber 1436 to pull cell1432 toward aperture 1434, forming a seal between the cell and aperture.Sealing and rupture of the cell membrane (whole cell entry) make theinside of cell 1432 electrically continuous with internal chamber 1436.In other embodiments, the membrane may be left unruptured butperforated, for example, by addition of channel-forming agents tointernal chamber 1436, or the membrane may be left unruptured andunperforated.

Electrical measurements then may be obtained. External chamber 1438 maybe connected to ground, while internal chamber 1436 may carry arecording electrode, generally connected to an amplifier. Ions passingthrough the membrane of cell 1432 create a current that may be measuredfollowing amplification with the amplifier. Device 1430 may be used tomeasure changes in ion channel-associated and/or transporter-associatedcurrents in the presence of potential drug candidates or othermodulators.

Multi-Aperture Embodiment

FIG. 60 shows a microfluidic device 1450 that is a multiplexed versionof device 1430, in accordance with aspects of the invention. Device 1450may include a shared internal chamber 1452 that extends around theperimeter of device 1450. Internal chamber 1452 may connect to a sharedexternal chamber 1454 using a plurality of apertures 1456, in this case,four. Each aperture may be isolatable, both electrically andfluidically, using control valves 1458 (V_(N), V_(S), V_(E), and V_(W)).In addition, each aperture may be disposed immediately adjacent a cellretention mechanism, such as retention site or trap 1460. Traps 1460 maybe arranged so as to facilitate parallel loading from a singlesuspension of cells (one reservoir) or from plural suspensions of cells(plural reservoirs). Internal chamber 1452 may be connected to a vacuumsupply, and a recording electrode and ground may be connected toexternal and internal chambers, 1452 and 1454, respectively.

Device 1450 may be readied and used as follows. First, internal chamber1452 may be loaded with internal buffer at internal-chamber port 1462(Port I), so that internal buffer is loaded up to apertures 1456. Next,open valves V_(N), V_(S), V_(E), and V_(W) may be closed, and cells maybe loaded as a suspension using an input mechanism at a common inputport 1464 (Port C). Then, the cell suspension may flow from Port C tooutput reservoirs 1466 (“outlet”). Single cells may be positioned andretained at each trap 1460 (N, S, E, W) using any suitable positioningand retention mechanisms, such as those described elsewhere in thisDetailed Description, for example, Examples 1-3. Once a desired numberof cells are retained by retention mechanisms, device 1450 may be usedfor cell analysis. The vacuum supply may be turned on, and one or morevalves at a time may be opened to form an electrical connection betweenthe internal and external chambers, through the corresponding aperture1456. The resistance of the connection may be used to determine if asufficient seal has been produced at the aperture, with the membrane ofthe retained cell. If so, recording may be commenced.

Device 1450 may be modified in any suitable fashion, incorporating anysuitable microfluidic mechanisms, such as those described in thisDetailed Description. For example, device 1450 may be structured to loadcells serially and/or in parallel, as described above in Examples 3-5.Furthermore, device 1450 may be included in an array of such devices toform a microfluidic array. Alternatively, or in addition, device 1450may include a perfusion mechanism, such as that described in Examples 2and 8, to allow precise delivery of selected reagents, to individualcells or to a plurality of cells, serially or in parallel. Similarly,device 1450 may measure electrical parameters of cells serially, thatis, by using one aperture at a time, or in parallel, by using two ormore apertures at a time, to obtain a summed reading of all connectedapertures.

FIG. 61 shows data from a simple statistical analysis illustrating a fewof the advantage of a multiplexed patch-clamp system, such as system1450. The fractional probability of successfully obtaining a seal in awell containing n apertures, P_(n), is related to the fractionalprobability of failed seal formation, P_(f), at a single aperture by theequation P_(n)=1−P_(f) ^(n). The probability of successful sealformation for a single aperture, P_(s), is related to P_(f) by theequation P_(f)+P_(s)=1. Therefore, if a seal is obtained successfully in50% of attempts, then with 4 apertures, P₄=1−(0.5)⁴=1−0.0625=0.9375.This corresponds to a 93.75% chance of obtaining at least one seal amongthe four apertures. FIG. 61 graphs the relationship between n (x-axis)and P_(s) (y-axis), with curve 1474 indicating (n, P_(s)) pairs thatgive a 95% probability of at least one of the n apertures forming asuccessful seal. (Apertures are called “channels” in FIG. 61.) P_(n)approaches unity, as P_(s) and/or n are increased.

Example 13 Multilayer Mold-Fabrication Method of Varying Height and/orCross-Sectional Geometries of Molded Microfluidic Structures

This example describes a method for producing, by soft lithography,microfluidic devices in which the cross-sectional geometry and/or heightof structures within and/or between microfluidic networks vary; seeFIGS. 62-71.

Background

A microfluidic network may include structures having a variety offunctions. For example, regulatable channels may include deflectablevalves, acting to partially or completely close the channels and/or topropel fluid through the channels. These channels generally are formedwith a semicircular or arcuate cross-sectional geometry to enableefficient valve closure. By contrast, particle-positioning channels mayact primarily as conduits for particles carried by fluid. Theseparticle-positioning channels generally have a height sufficient toallow particle movement. Accordingly, particle-positioning channels maybenefit from a rectangular cross section to enable particles to moveunrestrictedly from side-to-side (transversely) within the channels.Such unrestricted movement may allow particles to occupy a greaterproportion of the width of the channels, rather than just the centralportion, as with arcuate channels. Other channels may be size-selectiveor particle-restrictive, preventing entry of particles greater than agiven size. These particle-restrictive channels may have a height thatis less than the diameter of particles of interest. Furthermore,microfluidic networks may include cell/culture chambers with roofheights that are greater than more narrow channels, as described inExample 10, to improve the functionality of the chambers. Therefore,these and other structures described elsewhere in this DetailedDescription may benefit from, or require, roof height to vary in orderto function properly.

Single-layer molds often are formed using a desired thickness ofphotoresist on a substrate. The photoresist is patterned using acorresponding template that allows selective light exposure andphotosensitization of patterned regions of the photoresist. Depending onwhether the photoresist is positive or negative, the selectively exposedregions are either resistant or sensitive, respectively, to subsequentremoval during development with a suitable developing agent. Thisdevelopment nonspecifically removes all sensitive regions, generallydown to the substrate. The resistant regions are generally rectangularin cross-section, but may be heated to round their edges into anrounded/arcuate configuration. Accordingly, these remaining regions ofthe resulting mold may produce microfluidic channels of complementarystructure using soft lithography. In other embodiments, multiple layersof photoresist may be built up by sequential coating, masking, and

Despite the importance of varying height and/or cross-sectional shapeacross a microfluidic network, molds formed from a single layer ofselectively removable material, such as photoresist, may not allowsufficient flexibility in the structure of a microfluidic network formedfrom the mold. For example, the depth to which the single layer may beremoved cannot be varied readily, producing features of a single height,generally equal to the thickness of the single layer. Similarly,cross-sectional geometry may be difficult to vary within a single layerof the mold. Treatments that alter cross-sectional geometry, such asheating, also may act nonselectively across the single layer. Therefore,a method is needed for forming a mold using plural selectively removablelayers.

Description of Method

The method described in this example may be used to form channels withdifferent cross-sectional geometries and/or heights at distinctpositions within a microfluidic network. A mold is fabricated usingplural layers of photoresist that are each individually patterned,selectively removed according to the pattern, and optionally rounded byheating. Thus, each of the plural layers may contribute only a subset ofthe resulting mold, so that the mold's relief pattern is the sum of theremaining portions from each of the plural layers. Using the mold toform a microfluidic network allows various types of channels or otherpassages to be formed. Channels with a rounded/arcuate cross-sectionalshape may be formed in sections of the network where valves are needed.These sections may be connected with other portions of the network thatare formed to have a rectangular profile, to promote particle movementand to enable precise delivery of one or more particles to a specificarea of a microfluidic network. The specific area can be as small as thedimension of a single particle, such as a cell. These structures andother suitable microfluidic structures may be produced using the methoddescribed below. This method focuses on formation of a fluid layer, butmay be suitable for any portion(s) of a microfluidic system, including acontrol layer or a base layer (see Example 11).

A fluid-layer mold is fabricated in a first series of steps bymicromachining techniques. The fluid-layer mold may be used subsequentlyin a second series of steps, as described below, to mold a complementarymicrofluidic layer by soft lithography. FIGS. 62-68 illustrate howfluid-layer mold 1480 may be formed by sequentially disposing,patterning, and selectively removing three layers of photoresist on orabove a silicon wafer. Each layer is formed at a desired thickness byapplying the photoresist, and then rotating the wafer according to adefined rotational profile to produce the structure of FIGS. 62, 64, and67. Next, the photoresist is baked, patterned by exposure to UV light,and then developed to selectively remove portions of each layer, shownin FIGS. 63, 65, and 68. To mold closable channels, a photoresist layermay be baked at high temperature to round remaining portions, shown inFIG. 66. Each individual step is detailed further below.

The first layer may be applied directly to a bare silicon wafer (thesubstrate). The first layer may have any suitable thickness, in thiscase 5 μm, and may be formed with any suitable material, such as anegative photoresist, SU8 2005 (Microchem, Newton, Mass.). Afterapplication of the negative photoresist, the wafer may be rotatedaccording to a suitable rotational protocol to achieve a desiredthickness and consistency. For example, the wafer may be rotated asfollows: rotate to 500 rpm over 5 sec, maintain at 500 rpm for 5 sec,ramp to 3000 rpm over 8 sec, and then maintain at this speed for 30 sec.Then the rotation may be halted and the wafer heated according to asuitable heating protocol. For example, the wafer may be heated for 1min at 65° C., 2 min at 95° C., and finally 30 sec at 65° C. Thisheating process may drive off the solvent in which the photoresist maybe supplied. FIG. 62 shows mold 1480 with substrate 1482 carrying firstlayer 1484. The relative sizes of components here and in related FIGS.63-69 are not drawn to scale.

The first layer may be patterned and selectively removed as follows. Adesired template may be positioned in contact with the first layer andthen exposed to UV light, 160 J/cm². Next, the substrate/first layer maybe subjected to a suitable post-exposure heating protocol, such as: 1min at 65° C., 2 min 30 sec at 95° C., and 30 sec at 65° C.Unpolymerized (unexposed) first layer may be washed away with anysuitable developer, such as that supplied by Microchem, followed bywashing with acetone and then isopropanol. Then, the first layer may besubjected to a suitable post-development heating protocol, such as 1 minat 65° C., 5 min at 95° C., and then 30 sec at 65° C. This heatingprotocol may be followed by a post-development exposure with UV light,400 J/cm². FIG. 63 shows mold 1480 with first layer 1484 contributingfirst-layer relief-structure 1486 (residual first layer), which may havea height of 5 μm.

The second layer may be added next and may have any suitable thickness,in this case a thickness of 20 μm formed by spin coating. First, mold1480 may be treated with hexamethyldisilazane (HMDS) for 10 min. Next, asuitable patternable material, such as a positive photoresist, PLP 100(AZ Electronic Materials/Clariant Corporation) may be applied.Application may be by spin coating, using any suitable protocol, such asthe following: spin the wafer at 500 rpm, dispense the positivephotoresist to the wafer/residual first layer over 14 sec, spin 15 sec,ramp to 2000 rpm over 5 sec, and maintain at this speed for 30 sec.Rotation then may be stopped, and the second layer may be baked for 2min at 100° C. FIG. 64 shows mold 1480, at this intermediate stage,carrying second layer 1488, which covers first-layer relief-structure1486.

The second layer may be patterned and selectively removed as follows.Any suitable template may be positioned in contact with the second layerand exposed to UV light, 450 J/cm². Next, the second layer may bedeveloped (selectively removed) by any suitable protocol, such as 3 min.in AZ 400K 1/3 with deionized water. FIG. 65 shows mold 1480 afterpatterned removal of both first and second layers 1484, 1488.First-layer relief-structure 1486 and a second-layer relief-structure1490 may have distinct heights based on the thickness of photoresistfrom which they are formed.

Second-layer relief-structure 1490 may be rounded by any suitableheating protocol. For example structure 1490 may be rounded by thefollowing heating protocol: ramp from 70° C. to 100° C. (1° C./min),maintain 60 min at 100° C., ramp to 200° C. (1° C./min), maintain 60 minat 200° C., and ramp down to 40° C. (1° C./min). FIG. 66 shows how thisheating protocol may convert rectangular second-layer relief-structure1490 (FIG. 65) to rounded second-layer relief-structure 1492.

A third layer may be added next and may have any suitable thickness, forexample, a thickness of 20 μm. A suitable selectively removablematerial, such as negative photoresist SU8 2050 (Microchem), may beapplied to the wafer carrying the residual first and second layers. Spincoating may be achieved by the following protocol: the wafer is rampedto 500 rpm over 5 sec, maintained at this speed for 5 sec, ramped to5000 rpm over 17 sec, and maintained at this higher speed for 30 sec.The rotation is stopped. Next, the third layer may be heated by anysuitable, such as: 2 min. at 65° C., 3 min. at 95° C., and 30 sec at 65°C. FIG. 67 shows third layer 1494, which covers first-layer andsecond-layer relief-structures 1486, 1492 at this stage.

The third layer may be patterned and selectively removed as follows. Adesired template may be positioned in contact with the third layer andexposed to UV light, 310 J/cm². The exposed layer may be heated by anysuitable protocol, such as 1 min. at 65° C., 4 min. at 95° C., and 30sec at 65° C. Next, the third layer may be selectively removed with asuitable developer, such as that of Microchem, and then may be washedwith acetone followed by isopropanol. Subsequently, the third layer maybe subjected to a suitable post-development heating protocol, such as 1min. at 65° C., 5 min. at 95° C., and 30 sec at 65° C. Finally, thethird layer may be exposed to UV light in a post-development exposure of500 J/cm². FIG. 68 shows mold 1480 having a third-layer relief-structure1496.

Any suitable aspects of the method described above may be modified, andany patternable, selectively removable material may be used. Inaddition, any suitable number of layers may be used. Furthermore, eachlayer may have any desired thickness, according to the height of adesired relief structure. When optically patternable layers are used,each layer may be negative or positive photoresist, and may be used toform a rectangular or rounded cross-sectional profile. Relief structuresformed by distinct layers may be nonoverlapping, partially overlapping,and/or completely overlapping in specific regions or all regions of themold. Accordingly, relief structures may represent the sum of pluralselectively removed layers.

An exemplary method for forming a control-layer mold is as follows. Themold may be fabricated from a single layer of positive photoresist. A20-μm layer of suitable photoresist, such as positive photoresist PLP100, may be applied, patterned, selectively removed, and rounded asdescribed above for the second layer of the fluid-layer mold.

The fluid-layer and control-layer molds fabricated above may be used tomold a microfluidic chip using any suitable material, particularly anelastomeric material, such as polydimethylsiloxane (PDMS). ExemplaryPDMS elastomers are General Electric Silicones RTV 615, produced from atwo-component mixture of a prepolymer/catalyst and a crosslinker. Inthis two-component mixture, the prepolymer/catalyst (component A) is apolydimethylsiloxane bearing vinyl groups and a platinum catalyst, andthe crosslinker (component B) bears silicon hydride (Si—H) groups. Usingthese specific components, components A and B may function optimally ata ratio of about 10:1 (A:B). However, “off-ratios” above and below thisratio may be used for the fluid-layer membrane and the control layer topromote subsequent bonding. For example, the control layer may be formedat a ratio of about 4:1, to provide rigidity and thus mechanicalstability, and the fluid-layer membrane at a ratio of about 30:1. Theexcess of either component A or B in these two layers remain reactivenear the membrane surface. Accordingly, these two layers may be abuttedand bonded by post-curing with baking to fuse these layers into amonolithic structure (see below).

The fluid-layer and control-layer molds may be fabricated and joined asfollows. After treatment with trichloromethylsilane (TCMS), a relativelythin PDMS membrane, for example, about 50-150 μm, may be spun oncompleted fluid-layer mold 1480. FIG. 69 shows a membrane 1498 beingformed on fluid-layer mold 1480. In addition, a thicker PDMS layer, forexample, approximately 5-10 mm, may be formed on the control-layer mold.After suitable first-step curing, such as 90 min at 80° C., the controllayer may be detached from the mold, cut, and punched to interfaceproperly with control lines of the control layer. Then, this controllayer may be aligned with the fluid layer, while the fluid-layermembrane 1498 is still attached to the fluid-layer mold. Once assembled,the fluid and control layers may be cured a second time to chemicallybond them, using a post-curing step of heating for about 3 hours at 80°C. After post-curing, the resulting chip may be detached from thefluid-layer mold, cut, and punched to create fluid reservoirs thatinterface at desired positions with channels. Finally, the chip may bebonded to a suitable substrate, such as a glass cover slip, to completethe fluid channels.

The post-curing step may be modified to enhance compatibility withcells. Lower ratios of PDMS components A and B, such as 4:1 (A:B), tendto be toxic to cells, particularly during cell culture. This toxicitymay be due to a diffusible, toxic material(s) in the control layer.Thus, when a much thicker control layer, formed at a ratio of 4:1, isfused to a thin fluid-layer membrane, formed at a ratio of 30:1, theresulting monolithic structure may have the toxic characteristics of a4:1 layer, even within the fluid-layer portion. However, suitabletreatment of the control layer, either alone in contact with the fluidlayer membrane, reduces or eliminates this toxic characteristic.Suitable treatments that remove or modify the toxic material may includeexposure to heat, a chemical (such as a gas, a liquid, a plasma, etc.),radiation, light, and/or the like. (Such treatments also may reduce themovement of fluids within the channel, or components thereof, into thechip.) In some embodiments, longer post-curing at elevated temperaturemay remove or modify the toxic material(s), enhancing the effectivenessof the resulting chips for cell experiments. Such a longer post-curingstep may be conducted for about 6 hours, 12 hours, or more preferablyabout 24 hours or more at about 80° C.

Images of Molds and Chips

FIGS. 70 and 71 show photographic images of fluid-layer molds and thecorresponding microfluidic chips formed with these molds. Themicrofluidic networks represented here, have been shown and described insystem 1340 of Example 11 (FIG. 70) and in a modified form in system 850of Example 7 (FIG. 71). Distinct regions of each mold and fluid layerare indicated by letters A, B, and C. Area A corresponds to roundedsecond-layer relief-structures 1492 described above. These areas arecolor-coded in blue on many of the figures presented above. Channels ofarea A are about 200 μm wide and approximately 20 μm high. Area A may beused to form valves and pumps by overlapping control lines from acontrol layer with this area, such as valve 1500 in FIG. 71. Area Bcorresponds to third-layer relief-structure 1496. These areas arecolor-coded in red on many of the figures presented above. Channels ofarea B have a rectangular profile, approximately 100 μm wide and 20 μmhigh. These channels enable precise particle control, because they allowparticles to distribute across the width of the channel, following thewalls and/or the center of a fluid stream(s). Such channels may be usedto drive particles to precise areas of each chip. Area C corresponds tofirst-layer relief structure 1486. These areas are color-coded inturquoise on several of the figures presented above. These channels havea rectangular profile, 10 μm wide and 5 μm high. Small channels of thistype are used in combination with channels of area A or B to trap cellsor beads. Fluid may flow in these channels entry of cells or beads maybe restricted.

Example 14 Detection System for Kinetic Analyses in Microfluidic Systems

This example describes a detection system, including amodulation-demodulation method and the use of tracer materials, foranalysis of kinetic reactions involving particles in microfluidicsystems; see FIGS. 71A-F.

Background

Microfluidic systems may be used to measure the kinetics of many aspectsof cellular metabolism. However, metabolic processes of physiologicalsignificance can occur at substantially different rates, withcharacteristic times that may range from microseconds (10⁻⁶ sec) or lessto days (10⁵ sec) or more. Therefore, detection methods are needed tomeasure cellular events that occur at these vastly differing rates.

Time-resolved fluorescence spectroscopy has been one of the most popularapproaches to cellular kinetics studies. Typically, dye molecules areintroduced into cells, and emission from the molecules is produced byexcitation with an intense light source (such as an arc lamp or laser).The intensity of this emission is monitored over the course of theanalysis to infer the kinetics of a process under study. However, theemission intensity of the dye molecules may be reduced or extinguishedover time by photobleaching. As a result, some cellular processes thatoccur over relatively longer time periods may be more difficult tomonitor in a microfluidic system due to this photobleaching.

Because the rate of photobleaching is related to the intensity ofexciting light, a weaker light source may be used to reduce this rate.For example, FIG. 71A shows a comparison of photobleaching rates versustime using a relatively stronger laser (1.6 mW) and a relatively weakerlaser (1.6 μW). However, the exciting light source produces a reducedemission signal and signal-to-noise ratio, since the emission signal isproportional to the illumination intensity. Therefore, microfluidicanalyses would benefit from a detection system that reducesphotobleaching, increases the ratio of signal-to-noise, and/or allowskinetic analysis of both fast and slow processes.

Description of Detection System

This example describes an exemplary detection system for use withmicrofluidic assays, in accordance with aspects of the invention. Thedetection system may include a modulation-demodulation mechanism; seeFIGS. 71B-71E. This mechanism may improve signal-to-noise ratios,allowing use of weaker light sources, and/or reduce photobleaching,allowing use of stronger light sources. The detection system also mayinclude a method using tracer dyes to measure initiation of rapidkinetic reactions with particles; see FIG. 71F.

Light Detection Device

FIG. 71B shows an exemplary system 2010 for detecting an optical signalfrom a sample. System 2010 may include a light source 2012, optics 2014,a detector 2016, a digital storage device 2018, and amodulation-demodulation mechanism 2020.

Light source 2012 may be used to illuminate one or more particles withlight to visualize the particle and/or to perform an assay. The lightsource may generally may include any mechanism for producing lighthaving the desired characteristics, including time-dependent and/orcontinuous light sources. Suitable examples may include a laser, alight-emitting diode (LED), or a lamp, among others.

Optics 2014 may be used to receive light from light source 2012 anddirect the light at the particles and/or to receive light from theparticles and direct it to detector 2016. Optics may mediate anysuitable alteration of light to facilitate analysis, includingrefraction, reflection, diffraction, polarization, attenuation, spectralalteration, and/or scattering, among others. Suitable optics may includelenses, mirrors, fiber optics, filters, gratings, etalons, and/or thelike. Exemplary optics may include a conventional microscope or othersuitable optical device that is separate from, or partially or whollyintegrated with, a microfluidic system.

Modulation-demodulation mechanism 2020 may include a modulator 2022and/or a demodulator 2024. Modulator 2022 generally comprises anymechanism to provide time-dependent variation in the intensity ofexposure of sample to source 2012. This variation may be intrinsicand/or extrinsic to the light source. Intrinsic modulation occurs whenthe light source itself changes in intensity, as with a pulsed or strobelaser (such as a diode laser). Such a pulsed laser may be pulsed veryrapidly, up to millions of pulses per second, allowing forhigh-frequency illumination of particles. Extrinsic modulation occurswhen the light source is continuous (or quasi-continuous), but adownstream mechanism alters the intensity of light before it is incidenton the sample. Suitable extrinsic modulators include optical chopperwheels, Pockels cells, Kerr cells, acousto-optic modulators, and/orelectro-acoustic and other modulation devices. By contrast, demodulatorsgenerally comprise any mechanism for interpreting signals from detector2016 based on the activity of the modulator. The control and interplaybetween the modulator and demodulator may be performed using anysuitable mechanism, such as lock-in amplification using custom-designedand/or commercial devices.

Detector 2016 may be used to detect light, rapidly and/or repeatedly,and convert the detected light into representative electrical signals.Such a detector may include a photomultiplier tube, avalanchephotodiode, and/or other photodetector that provides the ability torapidly detect light signals produced by a source 2012 illuminating theparticles. Collecting light emitted through optical filters intophotomultiplier tubes or other photodetectors may enable conversion ofphotons to electrons for collection of quantitative information.

Digital storage device 2018 may digitize and/or store electrical signalsreceived from detector 2016. These stored signals may be retrieved,corrected, and/or otherwise converted or manipulated, and printed ordisplayed, as desired.

Exemplary Results Using a Modulation-Demodulation Mechanism forMicrofluidic Analysis

FIG. 71C shows a comparison of signal-to-noise ratios over time without(top) and with (bottom) source and signal modulation-demodulation. Inthis example, an embodiment of modulation-demodulation mechanism 2020boosts the signal-to-noise ratio by a factor of over 2000-fold.Accordingly, weaker light sources may be used and an emittedfluorescence signal may be measured over a longer time course.

FIG. 71D shows use of an embodiment of mechanism 2020 to determine therate at which a reagent-particle interaction occurs in a singleexperiment. Here, a biotinylated bead has been loaded into a trap on amicrofluidic chip, such as a chip designed according to system 250 ofExample 2. Dye-labeled streptavidin (reagent) is exposed to the bead ina pulsatile fashion, using cycles of staining and washing controlled byautomated operation of control valves. In this case, each ten-secondcycle includes a two-second exposure to reagent, followed by aneight-second exposure to wash buffer. Each cycle produces a spike influorescence intensity. However, the average fluorescence intensityachieves a near-maximal level in about twenty cycles. Accordingly,maximal staining occurred in about forty seconds (twenty cycles timestwo seconds per cycle). Therefore, flow-based exposure and washing maybe optimized to avoid time- and labor-intensive labeling and washingsteps, and to minimize use of reagent. The pulsatile exposureillustrated here may be used with any suitable particle and dyecombination to measure the rate at which interaction occurs.

FIG. 71E shows the ability of an embodiment of the microfluidicdetection system to measure a kinetic response of signal transduction ina cell. A calcium sensor dye, Fluo-3, was loaded into a cell, and thecell was trapped in a microfluidic chip, such as a chip designedaccording to system 250 of Example 2. The trapped cell was stimulatedwith ionomycin, at about time=120 sec, to promote release ofintracellular calcium. The graph shows intensity of fluorescence,corresponding to intracellular calcium concentrations, versus time. Suchan analysis measures the response of an individual cell, so compensatoryoscillations in calcium levels are visible.

Method Using Tracer Dyes

Most rapid reactions or events are difficult or impossible to measureunless their starting points can be precisely defined. Accordingly, atracer material, such as a tracer dye, may be included in a reagent ofinterest to indicate the time at which fluid containing the tracer dyeand reagent contacts a particle(s). Thus, first detection of the tracerdye in contact with the particle defines a zero time point at which areaction or event was initiated.

The tracer dye may have any optically detectable property and may beinert or reactive. Suitable optically detectable properties aredescribed above in Section VIII. Inert dyes generally do not contributedirectly to a detected assay result. Therefore, inert dyes generally donot affect cellular metabolism, and may not interfere optically orchemically with reagent dyes used to measure information aboutparticles. Inert dyes may be nonbinding or binding. Nonbinding dyes donot bind to particles and may simply mark fluid volumes. Binding dyesmay bind to particles, but do not contribute directly to a detectedresult from particles. By contrast, reactive dyes react with particlesand contribute to a detected result. Suitable reactive dyes may bedetectable when first combined with particles, but may show a change inan optical property during an assay. Inert or reactive dyes may beexcluded from cells, may partition into particles, or may be transportedinto the interior of cells. Inert and reactive dyes that may be suitableare sold by Molecular Probes, Eugene, Oreg.

Rapid perfusion mechanisms, such as perfusion mechanism 268 of Example 2above, coupled with a tracer dye and detection system described in thisexample, may allow very rapid analyses to be performed on particles.Such rapid analyses may measure events that occur in less than about 2sec, 1 sec, or 500 msec. Furthermore, these rapid analyses may beperformed on living cells to measure cell responses that are notdetectable readily by other methods.

FIG. 71F shows use of an embodiment of modulation-demodulation mechanism2020 and a tracer dye in a microfluidic system to measure the rate atwhich reagent is exposed to particles. A perfusion mechanism, such asmechanism 268, was used to expose a retention site to a fluorescent dye.The resulting increase in fluorescence was measured over time. At time“T,” an electrical signal was sent to a valve controller. After a shortmechanical delay of about 5 msec, fluorescence measured at the retentionsite begins to increase, reaching a maximum value in less than 100milliseconds. Accordingly, rapid kinetic analyses on a millisecond timescale may be performed using microfluidic systems described herein.

Example 15 Microfluidic Analysis of a Heterogeneous ParticlePopulation—Part I

This example describes microfluidic systems for sorting and analyzingheterogeneous populations of particles, particularly cells, based ondifferences in particle size; see FIG. 72.

Background

Heterogeneous cell populations, such as blood, present a challenge forrapid analysis. Cells of interest in blood generally need to beseparated from other cells that are of less interest to avoidinterference from these other cells. Accordingly, blood may need to betreated/manipulated to selectively lyse, coagulate, pellet, bind, and/ormodify, among others, specific cells within the blood. Suchmanipulations add to the time and expense required for analysis ofblood, because they involve trained personnel, expensive equipment,lengthy incubations, repeated transfer of relatively large volumes ofreagent or sample, and/or the like. In addition, such manipulationsexpose personnel to increased risk of exposure to infectious agents inthe blood. As a result, many diagnostic procedures using whole blood areexpensive and slow. Therefore, integrated systems are needed thatautomatically sort and analyze heterogeneous cell populations on amicrofluidic scale.

Description

This example describes microfluidic systems that sorts blood cells andother heterogeneous particle populations according to diameters ofindividual particles. With these systems very small volumes of blood maybe sufficient for statistically significant diagnoses or prognoses. Suchsystems may facilitate analysis of patient samples with improved speed,accuracy, safety, and/or cost, among others.

FIG. 72 shows a microfluidic system 1520 sorting cells. System 1520 isbased on system 250 of Example 2 and includes positioning and retentionmechanisms 264, 266 described in that example. A blood sample wasintroduced into system 1520 and directed toward retention chamber 270.Cells 1522 of this sample include red blood cells and platelets, but donot include detectable white blood cells, which would be retained by theretention mechanism due to their larger diameters. Cells 1522 enterchamber 270 but exit through size-selective side-wall channels 300.FIGS. 72A-D show time-lapse video images that include cells in chamber270 and in channels 300. White blood cells such as lymphocytes,monocytes, and granulocytes (neutrophils, eosinophils, and basophils),when present, would be retained in chamber 270. These white blood cellsare too large to pass through channels 300. Therefore, system 1520 maybe used to separate red blood cells and platelets from white bloodcells, for selective analysis of the white blood cells (or red bloodcells) in the system.

System 1520 may be modified to select plural populations of particles ofdifferent size. For example, the system may be modified to include aserial set of retention mechanisms. Outflow through size-selectivechannels 300 for each retention mechanism 270 may be directed partiallyor completely toward an input site of a successive retention mechanism.Each successive mechanism may have a reduced diameter of channel 300, sothat a reduced diameter of particle is retained in each successivemechanism. With this arrangement, larger particles are retained earlierin the series of mechanisms, whereas smaller particles are retainedlater in the series. Any suitable retention mechanism may be used ateach position in the series.

Particles retained in the retention mechanism of system 1520 or relatedsystems may be treated and analyzed. Particles may be treated byexposing them to desired reagents, for example, using perfusionmechanism 268 of Example 2, or by introducing reagents from any otherreservoirs included in system 1520. Thus, particles retained in distinctretention mechanisms may be isolated and exposed to distinct reagents,as described in Example 4. Systems such as system 1520 may enableon-chip staining and washing, eliminating any need for multiple pipetingand/or centrifugation steps during manipulation and detection.

Suitable characteristics of retained particles may be detected by flowor scanning cytometry, among others. In flow cytometry, particles aredetected while flowing past a detection mechanism, such as a lightsource coupled to a photodetector. Accordingly, particles may bereleased from each retention mechanism, for example, using a releasemechanism, such as described above in Example 7, to flow past adetector. Alternatively, or in addition, characteristics of particlesmay be detected or otherwise detected while the particles are relativelystationary, such as when localized in chamber 270. Photons may beconverted to electrons using photomultiplier tubes, avalanchephotodiodes, CCDs, or similar technologies. Light emitted from dyes maybe bright enough to detect using a single CCD, and scattered light mayyield enough structural information from particles, when combined withfunctional information, to identify specifically the type and state ofparticles.

Additional aspects of sorting a heterogeneous particle population aredescribed below in Example 26.

Example 16 Microfluidic Interaction of Specific Binding Pairs on Beads

This example describes detection of interaction between a specificbinding pair, biotin and avidin, on beads in a microfluidic system; seeFIGS. 73-74.

Background

Beads are used frequently by pharmaceutical and biotechnology companiesas carriers for drug targets, drug candidates, chemical syntheses,immunoassays, chromatography, and/or so on. However, small numbers ofbeads are difficult to manipulate, particularly to detect reactions thatoccur rapidly. As a result, using currently available technology, assayswith beads generally are conducted on a relatively large scale, wastingvaluable reagents and/or may measuring a reaction endpoint that missesvaluable earlier reaction information. Therefore, systems are needed tostudy interaction, including rapid interactions, using small numbers ofbeads.

A specific binding pair, biotin/streptavidin, was selected forinteraction on beads; see FIG. 73. Biotin is a vitamin with a molecularweight of 244 daltons. Its partner, avidin, binds biotin with fiercetenacity, being the strongest non-covalent attachment known, with anassociation constant of 10¹⁵ M⁻¹. This binding reaction has been studiedintensively for many decades, and there is a rich literature. The greatstrength of this binding suggests that it might be a good model systemfor the study of biological binding reactions in general. It has alsoformed the basis for many detection and signal amplification strategiesfor both research and clinical labs.

Avidin and streptavidin are vertebrate and bacterial biotin partners,respectively. Avidin is a protein with a molecular weight of about 68kilodaltons, including four identical subunit chains, each 128 aminoacids long. Avidin is found predominantly in the egg white of birds,amphibia, and reptiles. The protein streptavidin, produced by thebacterium Streptomyces avidinii, has a structure very similar to avidin,also binding biotin tightly. However, streptavidin often exhibits lowernonspecific binding, and thus is frequently used in place of avidin.

Method

Materials for measuring biotin/avidin interaction were as follows. Amicrofluidic chip was fabricated based on system 250 of Example 2.Beads, 6.7-micron biotinylated polystyrene microspheres, were obtainedfrom Spherotech Corporation. Other buffers and reagents includedphosphate-buffered saline (PBS) containing 0.5% BSA (sterile filtered),and the streptavidin conjugated fluorophores streptavidin-Alexa 350,streptavidin-Alexa 488, and streptavidin-PE (phycoerythryn), eachobtained from Molecular Probes. Binding reactions were monitored with aninverted fluorescent microscope connected to a video camera.

The analysis was conducted according to the following numbered steps.

The fluid network of the chip was washed with water, then withPBS/BSA/Tween-20.

Beads were captured on the chip using its retention chamber.

Streptavidin-conjugates were loaded into reagent-wells on the chip (2 μLof each conjugate in 1 mL PBS).

The captured beads were exposed to each of the conjugates.

A 63× oil-immersion lens on the inverted microscope was used to maximizefluorescent signal. Blue and green/red filter sets were used.

In some cases the rate of photobleaching by the detection mechanismexceeded the rate at which fluorescent conjugates were captured by thebeads. In these cases, the procedure was repeated without constantexposure to UV, opening the UV shutter only long enough to documentbinding.

Results

FIG. 74 shows the results of portions of the analysis as selected videoframes during exposure of streptavidin-Alexa 488 conjugate to retainedbeads. In FIG. 74A, the beads have been loaded in chamber 270, but havenot bound detectable amounts of the conjugate and are not detectable. InFIG. 74B, beads 1550 are detectable above background. In FIG. 74C, theyhave become readily detectable, after unbound conjugate is washed out ofthe chamber. FIG. 74D shows beads 1550 under bright field illuminationto localize the beads and demonstrate that all beads in the chamber arestained with conjugate.

Similar exposures to the other conjugates gave less intense staining.Detectable staining with streptavidin-Alexa 350 was visible, butstreptavidin-PE did not yield a detectable signal. However, moresensitive detection mechanisms, such as a laser scanning cytometer mayallow detection of streptavidin-PE binding.

Example 17 Measuring Ion Flux in Cells Using a Microfluidic System

This example describes analysis of intracellular ion concentrations,such as calcium ion concentrations, using a microfluidic system; seeFIG. 75.

Background

Calcium is a very important intracellular ion. It plays a vital role inthe transduction of signals from the cell membrane to the cell cytoplasmand nucleus. A change in intracellular calcium levels is an indicationthat the cell is responding to a stimulus. Many stimuli causemobilization of calcium, either as an influx from the extracellularmedium or by release from intracellular pools. Fluorescent calciumindicators allow this mobilization to be observed.

Method

Materials used for measuring intracellular calcium levels were asfollows. A microfluidic chip was constructed based on a modified versionof system 850 of Example 7. Fluo 3/AM, a fluorescent Ca⁺² indicator dyewas obtained from Calbiochem, and used as a 5 mM stock. Ionomycin, freeacid form, was also obtained from Calbiochem. Cells were Jurkat T-cellsand were grown in RPMI media.

The analysis was conducted according to the following numbered steps.

Cells were cultured in RPMI media.

Cells/media (5 mL) were pelleted at 1000 rpm for 5 min.

The cells were resuspended in RPMI containing 5 μM Fluo-3 (10 mL RPMIplus 8 μL FLUO-3 AM).

The cell/Fluo-3 mixture was incubated at 37° C. for 30 min to load thecells with indicator dye.

The cells were pelleted and washed twice with Hanks' balanced saltsolution (HBBS) containing 20 mM HEPES (200 μL 1M Hepes in 10 mL HBBS).

The cells were placed in the input reservoir of the chip.

The microscope and video camera were set up.

HBBS/Hepes buffer was pumped across cells, acting as a shield buffer toregulate exposure to reagent.

HBBS/Hepes containing ionomycin was pumped past the cells, but in alayer spaced from the cells by the shield buffer.

The flow of shield buffer flow was terminated, exposing the cells toionomycin.

Calcium flux was recorded with the video camera as ionomycin contactedthe cells.

Results

FIG. 75 shows the results of the analysis, as selected video frames,before and after exposure of Jurkat cells, loaded with indicator dye, toionomycin. FIG. 75A shows two cells 1570 captured in retention site 1572and visualized under bright field illumination. In FIG. 75B, these cellslack fluorescence before ionomycin exposure. In contrast, FIG. 75Creveals fluorescence (green signal) of cells 1570 very soon afterionomycin exposure. A negative control demonstrated that ionomycin wasrequired for this fluorescence (not shown).

Example 18 Microfluidic Analysis of Cell-Surface Markers

This example describes a method for detection of cell-surface markers,such as CD4 and CD8, on cultured T-cells using labeled antibodies.

Background

The CD4 molecule recognizes an antigen that interacts with class IImolecules of the major histocompatibility complex (MHC) and is theprimary receptor for the human immunodeficiency virus (HIV) (Dalgleishet al., 1984; Maddon et al., 1986). The cytoplasmic portion of theantigen is associated with the protein tyrosine kinase p56^(lck) (Ruddet al., 1989). The CD4 antigen may regulate the function of the CD3antigen/T-cell antigen receptor (TCR) complex (Kurrle et al., 1989). TheCD4 antibody reacts with monocytes/macrophages that have an antigendensity lower than that on helper/inducer T lymphocytes (Wood et al.,1983).

The CD8 antigen is present on the human suppressor/cytotoxicT-lymphocyte subset (Evans, et al., 1981; Ledbetter et al., 1981) aswell as on a subset of natural killer (NK) lymphocytes (Lanier et al.,1983). The CD8 antigenic determinant interacts with class I MHCmolecules, resulting in increased adhesion between the CD8⁺ Tlymphocytes and the target cells (Anderson et al., 1987; Eichmann etal., 1987; Gallagher et al., 1988). Binding of the CD8 antigen to classI MHC molecules enhances the activation of resting T lymphocytes. CD8recognizes an antigen expressed on the 32-kDa a-subunit of adisulfide-linked bimolecular complex (Moebius, 1989). The cytoplasmicdomain of the α-subunit of the CD8 antigen is associated with theprotein tyrosine kinase p56^(lck) (Rudd et al., 1989; Gallagher et al.,1989).

Determining the percentages of CD4+ and CD8+ lymphocytes may be usefulin monitoring the immune status of patients with immune deficiencydiseases, autoimmune diseases, or immune reactions. The relativepercentage of the CD4+ subset is depressed and the relative percentageof the CD8⁺ subset is elevated in many patients with congenital oracquired immune deficiencies such as severe combined immunodeficiency(SCID) and acquired immunodeficiency syndrome (AIDS) (Schmidt, 1989;Giorgi, 1990).

The percentage of suppressor/cytotoxic lymphocytes can be outside thenormal reference range in some autoimmune diseases (Antel et al., 1986)and in certain immune reactions such as acute graft-versus-host disease(GVHD) and transplant rejection (Gratama et al., 1984; Bishop et al.,1986). The relative percentage of the CD8⁺ lymphocyte population mayoften be decreased in active systemic lupus erythematosus (SLE) but canalso be increased in SLE patients undergoing steroid therapy(Wolde-Mariam et al., 1984).

The CD4⁺/CD8⁺ (helper/suppressor) lymphocyte ratio, quantified as theratio of CD4 fluorescein isothiocyanate (FITC)-positive lymphocytes toCD8 phycoerythrin (PE)-positive lymphocytes, has been used to evaluatethe immune status of patients with, or suspected of developing,autoimmune disorders or immune deficiencies (Antel et al., 1986;Wolde-Mariam et al., 1984; Smolen et al., 1982). In many cases, therelative percentages of helper lymphocytes decline and suppressorlymphocytes increase in immune deficiency states. These states may alsobe marked by T-cell lymphopenia (Ohno et al., 1988). In addition, theratio has been used to monitor bone marrow transplant patients for onsetof acute GVHD (Gratama et al., 1984).

The Jurkat cell, a human mature leukemic cell line, phenotypicallyresembles resting human T lymphocytes and has been widely used to studyT cell physiology. These cells are round, growing singly or in clumps insuspension. They were established from a human T cell leukemia in theperipheral blood of a 14-year-old boy with acute lymphoblastic leukemia(ALL) at first relapse in 1976. This cell line is also called “JM”(JURKAT and JM are derived from the same patient and are sister clones).Occasionally JM may be a subclone with somewhat divergent featuresconfirmed as human with IEF of AST, LDH, and NP. Jurkat cells have thefollowing general restriction properties: CD2+, CD3+, CD4+, CD5+, CD6+,CD7+, CD8−, CD13−, CD19−, CD34+, TCRalpha/beta+, and TCRgamma/delta−.

Method

Materials used for analysis of CD4 and CD8 were as follows. Microfluidicchips was constructed based on a modified version of system 850 ofExample 7. Jurkat T-cells were cultured in RPMI. Fluorophore-conjugatedantibodies, CD4-fluorescein isothiocyanate (FITC) and CD8-phycoerythryn(PE), were used. Buffer for dilution, focusing, washing, etc. was PBScontaining 0.5% BSA. Data were collected with an inverted fluorescentmicroscope equipped with a video camera.

The analysis was conducted according to the following numbered steps.

Jurkat cells were grown in RPMI and then pelleted (10 mL ofmedia/cells).

The cells were resuspended in 1 mL PBS containing 0.5% BSA.

Anti-CD4-FITC and anti-CD8-PE-antibody-conjugates were diluted 1:100 inPBS containing 0.5% BSA.

The chip was prepared by running deionized water through themicrofluidic network and then was mounted on an inverted fluorescentmicroscope. The 100× or 63× oil-immersion lens was used to maximizefluorescent signal.

Cells were loaded onto the chip, positioned, and retained.

The diluted antibody-conjugates were loaded into separate reagentinput-wells of the chip.

Exposure to light from the UV lamp was minimized to avoidphotobleaching.

Anti-CD4-FITC was exposed to cells for 2 min.

The valve regulating CD4 antibody-conjugate flow was closed.

The shield-buffer flow line was opened to remove unbound antibodies.

The UV excitation shutter was opened and cell fluorescence was recorded.

When fluorescence was dim or invisible, the UV shutter was closed andsteps 8 through 11 were repeated.

Step 12 was repeated until fluorescence was observed and documented.

As a negative control, steps 8 through 12 were repeated usinganti-CD8-PE.

Results

Anti-CD8 antibody-conjugate did not bind to Jurkat cells, and thereforelittle or no red fluorescence was visible in the time frame needed tovisualize the green fluorescence of the anti-CD4 antibody-conjugate. Theprocedure may be repeated with continuous UV exposure to observeantibody binding in real-time.

REFERENCES

Maddon P, Dalgleish A, McDougal J, Clapham P, Weiss R, Axel R. The T4gene encodes the AIDS virus receptor and is expressed in the immunesystem and the brain. Cell. 1986; 47:333-348.

Dalgleish A, Beverly P, Clapham P, Crawford D, Greaves M, Weiss R. TheCD4 (T4) antigen is an essential component of the receptor for the AIDSvirus. Nature. 1984; 312 (December):763-767.

Rudd C, Burgess K, Barber E, Schlossman S. Monoclonal antibodies to theCD4 and CD8 antigens precipitate variable amounts of CD4/CD8-associatedp56-lck activity. In: Knapp W, Dörken B, Gilks W R, et al, eds.Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: OxfordUniversity Press; 1989:326-327.

Kurrle R. Cluster report: CD3. In: Knapp W, Dörken B, Gilks W R, et al,eds. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford:Oxford University Press; 1989:290-293.

Wood G, Warner N, Warnke R. Anti-Leu-3/T4 antibodies react with cells ofmonocyte/macrophage and Langerhans lineage. J Immunol. 1983;131(1):212-216.

Evans R, Wall D, Platsoucas C, et al. Thymus-dependent membrane antigensin man: Inhibition of cell-mediated lympholysis by monoclonal antibodiesto the TH₂ antigen. Proc Natl Acad Sci USA. 1981; 78(1):544-548.

Ledbetter J A, Evans R L, Lipinski M, Cunningham-Rundles C, Good R A,Herzenberg L A. Evolutionary conservation of surface molecules thatdistinguish T lymphocyte helper/inducer and T cytotoxic/suppressorsubpopulations in mouse and man. J Exp Med. 1981; 153(February):310-323.

Lanier L L, Le A M, Phillips J H, Warner N L, Babcock G F.Subpopulations of human natural killer cells defined by expression ofthe Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol. 1983;131(4):1789-1796.

Anderson P, Blue M-L, Morimoto C, Schlossman S. Cross-linking of T3(CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes.J Immunol. 1987; 139:678-682.

Eichmann K, Johnson J, Falk I, Emmrich F. Effective activation ofresting mouse T lymphocytes by cross-linking submitogenic concentrationsof the T-cell antigen receptor with either Lyt-2 or L3T4. Eur J Immunol.1987; 17:643-650.

Gallagher P, Fazekas de St. Groth B, Miller J. CD4 and CD8 molecules canphysically associate with the same T-cell receptor. Proc Natl Acad SciUSA. 1989; 86:10044-10048.

Moebius U. Cluster report: CD8. In: Knapp W, Dörken B, Gilks W R, et al,eds. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford:Oxford University Press; 1989:342-343.

Bernard A, Boumsell L, Hill C. Joint report of the First InternationalWorkshop on Human Leucocyte Differentiation Antigens by theinvestigators of the participating laboratories: T2 protocol. In:Bernard A, Boumsell L, Dausett J, Milstein C, Schlossman S, eds.Leucocyte Typing. Berlin: Springer-Verlag; 1984:25-60.

Schmidt R. Monoclonal antibodies for diagnosis of immunodeficiencies.Blut. 1989; 59:200-206.

Centers for Disease Control. Guidelines for the performance of CD4⁺T-cell determinations in persons with human immunodeficiency virusinfection. MMWR. 1992; 41 (No. RR-8):1-17.

Giorgi J, Hultin L. Lymphocyte subset alterations and immunophenotypingby flow cytometry in HIV disease. Clin Immunol Newslett. 1990;10(4):55-61.

Antel J, Bania M, Noronha A, Neely S. Defective suppressor cell functionmediated by T8⁺ cell lines from patients with progressive multiplesclerosis. J Immunol. 1986; 137:3436-3439.

Gratama J, Naipal A, Oljans P, et al. T lymphocyte repopulation anddifferentiation after bone marrow transplantation: Early shifts in theratio between T4⁺ and T8⁺ T lymphocytes correlate with the occurrence ofacute graft-versus-host disease. Blood. 1984; 63(6):1416-1423.

Bishop G, Hall B, Duggin G, Horvath J, Sheil A, Tiller D.Immunopathology of renal allograft rejection analyzed with monoclonalantibodies to mononuclear cell markers. Kidney Internat. 1986;29:708-717.

Wolde-Mariam W, Peter J. Recent diagnostic advances in cellularimmunology. Diagnost Med. 1984; 7:25-32.

Smolen J, Chused T, Leiserson W, Reeves J, Ailing D, Steinberg A.Heterogeneity of immunoregulatory T-cell subsets in systemic lupuserythematosus: Correlation with clinical features. Am J Med. 1982;72:783-790.

Ohno T, Kanoh T, Suzuki T, et al. Comparative analysis of lymphocytephenotypes between carriers of human immunodeficiency virus (HIV) andadult patients with primary immunodeficiency using two-colorimmunofluorescence flow cytometry. J Exp Med. 1988; 154:157.

Example 19 Measuring Cell Lysis in a Microfluidic System

This example describes capture, lysis, and staining of cells.

Background

Acridine orange (AO) was used for staining. AO binds to single strandednucleic acids as a dimer, which fluoresces red in color, and to doublestranded nucleic acids as a monomer, which fluoresces green. Thisdifference in fluorescent wavelength is caused by differentialaccessibility of AO molecules to the nucleic acid binding sites. AOfluorescence is also pH sensitive, staining acidic organelles, such aslysosomes, orange.

Method

Materials used for measuring lysis were as follows. Microfluidic chipswas constructed based on system 250 of Example 2. Jurkat T-cells werecultured in RPMI. Acridine Orange was dissolved at 5 μg/ml in PBS.Solutions or liquids to lyse cells included PBS containing 0.05%hydrogen peroxide, deionized water, PBS containing 2% TWEEN 20 (0.2 μmfiltered), and WINDEX. Data were collected on an inverted fluorescentmicroscope equipped with a video camera.

The analysis was conducted according to the following numbered steps.

Jurkat cells were grown in RPMI and pelleted (10 mL of culturemedia/cells).

The cells were resuspended in 5 mL PBS containing 5 μg/ml AcridineOrange, or left unstained for use on a control chip. For the controlchip, proceed to step 5.

The cells were incubated 10 min at room temperature.

The cells were pelleted and washed twice in PBS.

The cells were resuspended in 1 mL PBS.

The chip was preparing by washing the microfluidic network withdeionized water, and then was mounted on an inverted fluorescentmicroscope. The microscope's 63× oil-immersion lens was used to maximizefluorescent signal.

The cells were loaded onto the chip, positioned, and retained.

PBS containing peroxide was loaded into a reagent-well of the chip.

Exposure of the chip to light from the UV lamp was minimized, tominimize photobleaching.

The UV shutter was opened to expose stained cells to fluorescent light.

PBS containing peroxide was pumped over the cells for 2 min or untillysis or photobleaching occurred.

Cells were then exposed sequentially to PBS/2% TWEEN-20, WINDEX, andfinally water.

Results

The conditions of peroxide, TWEEN, and WINDEX did not lyse the cells onthe first attempt of this experiment. Subsequently, water was usedsuccessfully to demonstrate cell lysis. Lysis probably occurred underthe other conditions, but was not as obvious. Jurkat cells are fairlyrobust and may not be a good model cell line for this experiment.

Example 20 Inducing and Detecting Cell Apoptosis in a MicrofluidicEnvironment

This example describes induction and detection of cell apoptosis in amicrofluidic system; see FIG. 76.

Background

Apoptosis, also termed programmed cell death, is a carefully regulatedprocess of cell death that Occurs as a normal part of development.Inappropriately regulated apoptosis is implicated in disease states,such as Alzheimer's disease and cancer. Apoptosis is distinguished fromnecrosis, or accidental cell death, by characteristic morphological andbiochemical changes, including compaction and fragmentation of thenuclear chromatin, shrinkage of the cytoplasm, and loss of membraneasymmetry.¹⁻⁵

Phosphatidylserine (PS) distribution also can act as a marker forapoptosis. In normal viable cells, phosphatidylserine is located on thecytoplasmic side of the cell membrane. However, in apoptotic cells, PSis translocated from the inner to the outer leaflet of the plasmamembrane, thus exposing PS to the cell exterior.⁶ In leukocyteapoptosis, PS on the outer surface of the cell marks the cell forrecognition and phagocytosis by macrophages.^(7,8) The humananticoagulant, annexin V, is a 35-36 kD Ca⁺²-dependentphospholipid-binding protein that has a high affinity for PS.⁹ Annexin Vcan identify apoptotic cells by binding to PS exposed on the outerleaflet.¹⁰ Bound annexin V may be detected through a dye, a specificbinding member conjugated to annexin V, an anti-annexin-V antibody,and/or the like.

Hydrogen peroxide has been shown to induce markers of apoptosis, such asPS translocation, in cultured cells. The cellular toxicity of hydrogenperoxide (H₂O₂) is initiated by oxidative stress, resulting in rapidmodification of cytoplasmic constituents, depletion of intracellularglutathione (GSH) and ATP, a decrease in NAD⁺ level, an increase in freecytosolic Ca²⁺, and lipid peroxidation.¹¹ H₂O₂ also activates themitochondria permeability transition pore and the release of cytochromec.¹² In the cytoplasm, cytochrome c, in combination with Apaf-1,activates caspase-9, leading to the activation of caspase-3 andsubsequent apoptosis¹³⁻¹⁵.

Method

This example demonstrates induction and detection of cell apoptosis in amicrofluidic system. Jurkat cells are positioned and retained in amicrofluidic system, and then programmed cell death is initiated byexposure of these cells to hydrogen peroxide. Translocation of PS to theouter membrane leaflet is monitored with annexin V, to measureapoptosis. At the same time, cells are exposed to propidium iodide,which stains cells with disrupted membranes, an indicator of necrosisrather than apoptosis.

Materials used were as follows. Microfluidic chips were constructedbased on system 250 of Example 2. Jurkat T-cells were cultured in RPMI.The VYBRANT Apoptosis Assay Kit #2 was obtained from Molecular Probes,Eugene, Oreg. This kit includes fluorophore-conjugated annexin V (green)and propidium iodide (red). Data were collected on an invertedfluorescent microscope equipped with a video camera.

The analysis was conducted according to the following numbered steps.

The video camera was turned on.

Cells were trapped in the retention chamber of the chip.

Annexin-V-conjugate was loaded into reagent well #1 of the chip.

Propidium iodide was loaded into reagent well #2 of the chip.

Binding Buffer (BB) was loaded into the shield buffer well of the chip.

The cells were perfused with BB for 5 min.

The cells were perfused with annexin-V-conjugate for 5 min.

Cells were checked for staining. (Note: This is a negative control. Nostaining occurred at this stage because the cells had not apoptosed.)

The valves regulating flow of the shield buffer and reagent wells wereeach closed.

The BB was replaced with 800 μM H₂O₂ in PBS.

The cells were exposed to the H₂O₂/PBS by opening the valve regulatingflow from of the shield buffer.

Cells were observed under light microscopy during induction ofapoptosis.

After 15 min, the valve regulating flow of the shield buffer was closed.The well was washed with BB, and then replaced with BB.

The cells were then perfused with BB for 5 min.

The valve for the annexin-V-conjugate was opened, and the shieldingbuffer valve was closed.

The cells were exposed to the annexin-V-conjugate for 5 min.

The valve controlling the annexin-V-conjugate was closed, and the BBvalve was opened to wash the cells.

The cells were exposed to excitation light by opening the microscopeshutter. Green fluorescence indicated a positive reaction forphosphatidylserine.

The valve that regulates flow of propidium iodide (“the PI valve”) wasopened, while the valve that regulates BB (“the BB valve”) was closed.

After 2 min, the BB valve was reopened, and the PI valve was closed.

After washing for 5 min, the fluorescent shutter was opened while usingthe red filter set on the microscope.

Finally, the BB was replaced with water, and the cells were lysed andthen re-exposed to the PI.

Results

FIG. 76 shows selected video frames from this analysis. In panel A,cells 1590 have been trapped in chamber 270 and are visible under brightfield illumination. Panels B and C compare labeling of cells with theannexin-V-conjugate before (B) and after (C) exposure to hydrogenperoxide. Cells 1590 do not label with the annexin-conjugate beforeexposure to hydrogen peroxide (panel B), but a weak annexin-conjugatesignal is detectable after hydrogen peroxide exposure (panel C),demonstrating that at least some of the cells have initiated apoptosis.Panels D-F compare propidium iodide staining of cells 1590 at differenttimes during the analysis. Panels D and E show no propidium iodidestaining, either before or after induction of apoptosis by exposure tohydrogen peroxide. In contrast, panel F reveals detectablepropidium-iodide staining after exposure of cells to water, whichrenders the cells necrotic.

REFERENCES

Immunol. Cell Biol. 76, 1 (1998).

Cytometry 27, 1 (1997).

J. Pharmacol Toxicol. Methods 37, 215 (1997).

FASEB J. 9, 1277 (1995).

Am J. Pathol. 146, 3 (1995).

Cytometry 31, 1 (1998).

J. Immunol. 148, 2207 (1992).

J. Immunol. 151, 4274 (1993).

J. Biol. Chem. 265, 4923 (1990).

Blood 84, 1415 (1994).

Am. J. Physiol. 273, G7 (1997).

Free Radic. Biol. Med. 24, 624 (1998).

FEBS Lett. 447, 274 (1999).

Cell 91, 479 (1997).

Annu. Rev. Cell Dev. Biol. 15, 269 (1999).

Example 21 Analysis of Aquatic Microorganisms in a Microfluidic System

This example describes the capture and visualization of aquaticmicroorganisms, such as plankton, using a microfluidic system.

Background

Plankton are a very diverse group of marine and fresh water organismsthat spend some or all of their lives drifting in water. Planktonrepresent both the animal and plant kingdoms and include a range ofsizes from submicron to over a centimeter. These seemingly listlessorganisms play critical roles, both positive and negative, in the healthof not only other aquatic organisms but also in the composition of theearth's atmosphere. For example, these organisms are thought to producea large fraction of the earth's oxygen. In addition, they play acritical role in global carbon dioxide exchange, removing much of theexcess carbon dioxide produced by burning fossil fuels and sending thiscarbon dioxide to the ocean floor. In contrast, some plankton areinfamous for their negative impact on the economy. For example,explosive population growth of dinoflagellate plankton produce a toxic“red tide” that poisons fish and shellfish. However, occurrences of redtides are difficult to predict and/or prevent, resulting in extensivefish-kills and beach closures, which have a large economic impact.Therefore, systems are needed to manipulate, treat, and analyzeplankton, including laboratory or natural populations that benefit orharm the environment.

Method and Results

This example provides a microfluidic system capable of manipulating anddetecting small plankton, particularly picoplankton (0-2 μm),ultraplankton (2-5 μm), and/or nannoplankton (5-60 μm). Plankton may beretained, treated, and/or detected in an integrated microfluidicenvironment.

Plankton were manipulated and detected in a microfluidic system asfollows. A sample of seawater was collected from San Francisco Bay andcentrifuged to concentrate organisms in the sample. A 20 μL aliquot ofthe concentrated sample was loaded into the input reservoir ofmicrofluidic system 250, described in Example 2 above.Naturally-fluorescent plankton were retained in chamber 270 and detectedsuccessfully by fluorescent microscopy (not shown).

This method of this example may be modified by changing any suitableparameters. For example, plankton may be collected from freshwatersources or cultured, an aqueous plankton sample may be loaded directlyinto a microfluidic environment without concentration, and/or retainedplankton may be exposed to any suitable reagents. Alternatively, or inaddition, microfluidic systems may be used that sort a heterogeneouspopulation of plankton according to a physical property (such as size ordensity, among others) or a measured property/characteristic (such aslabeling with a dye and/or specific binding member).

Example 22 Analysis of Membrane Trafficking in a Microfluidic SystemUsing Membrane Dyes

This example describes microfluidic analysis of membrane traffickingpathways in cells treated with membrane-labeling dyes.

Background

Studies of vesicle trafficking often rely on optically detectable dyesthat label membranes. Brief exposure of cells to such a dye results inlabeling of the surface-membrane of these cells. Subsequent dye movementto interior membranes, such as endosomes, Golgi apparatuses, lysosomes,and/or endoplasmic reticulum, tracks corresponding transit of surfacemembranes, receptors, and/or ligands, among others, throughintracellular vesicle trafficking pathways. Using this approach, cellendocytic, recycling, degradative, and/or secretory pathways may bemonitored and analyzed.

Some “FM” dyes available from Molecular Probes bind to cell membranes.Thus these FM membrane dyes may be used as general-purpose probes forendocytosis, because they are generally nontoxic. FM membrane dyes arevirtually non-fluorescent in aqueous solution, but become intenselyfluorescent upon association with a membrane.

Goals and Method

The goals of this analysis included the following. I) Define thestaining conditions for two FM membrane dyes, FM 1-43 and FM 4-64, usingJurkat cells. FIGS. 77 and 78 show the structure and excitation/emissionspectra of these dyes. These two FM dyes have substantiallynonoverlapping emission spectra. II) Test the affinity of FM dyes formicrofluidic chips formed with PDMS, to define a background level ofstaining. III) Trap a Jurkat cell in a microfluidic chip and performtwo-color staining of the cell using the two FM membrane dyes.

Materials used for this analysis included the following. FM 1-43 and FM4-64 were obtained from Molecular Probes. Microfluidic chips wereproduced based on system 250 of Example 2. Results were collected andrecording using an inverted fluorescent microscope equipped with a videocamera.

Conditions for labeling Jurkat cells with FM membrane dyes weredetermined with the following labeling protocol.

Cultured Jurkat cells (5 mL of cells/media) were pelleted bycentrifugation at 1000 rpm for 5 min.

The cell pellet was washed twice with PBS.

The cell pellet was resuspended in 2 mL PBS.

Aliquots (500 μL) of the resulting cell suspension were dispensed intofour microcentrifuge tubes.

Dye was added to each of the four tubes as follows: no dye was added totube #1, FM 1-43 was added to tube #2, FM 4-64 was added to tube #3, andboth FM 1-43 and FM 4-64 were added to tube #4. The final dyeconcentration for each dye was 2 μM.

The cells were observed with the fluorescent microscope.

Each staining condition was documented by saving digital image files.

Labeling of the microfluidic chip with the FM membrane dyes to determinebackground signal was carried out as follows.

Each dye was diluted to a final concentration of 2 μM in PBS.

FM 1-43 (5 μL) was introduced into a first chip.

FM 4-64 (5 μL) was introduced into a second available chip.

A mixture of the FM 1-43 and 4-64 dyes (1:1) was introduced into a thirdchip.

Each dye-loaded chip was observed using a fluorescent microscope.

The level of background staining was determined relative to fluorescenceintensity of the cells stained with FM dyes in part A above.

Cells were labeled with FM dyes in a microfluidic system as follows.

Unlabeled Jurkat cells were loaded and captured in a microfluidic chipusing PBS as a carrier buffer.

Each FM membrane dye (5 μL) was placed in one of the two reagent wellson the chip.

Chip features and cells were visualized using minimal incandescentlight.

The video camera was turned on, and the 100× oil-immersion objective onthe fluorescent scope was used.

The first FM membrane dye (1-43) was delivered to the cells.

The fluorescent signal was observed.

The second FM membrane dye (4-64) was delivered to the cells.

The fluorescent signal was observed.

Steps 5-8 were repeated as necessary until the signal intensity wasmaximized.

Results

The results of the three protocols are as follows.

Protocol A produced significant labeling of Jurkat cells with the dyesafter a 5-minute incubation at room temperature. Each dye stained thecells with sufficient intensity to visualize using the fluorescentmicroscope. For example, FIG. 79 shows Jurkat cells stained with FM1-43. However, the emission profile of each dye was not distinguishableas a discrete color using the green/red filter set on a Leicamicroscope. Properly selected filter sets may allow a two-color assayusing these dyes.

Protocol B produced significant background labeling of microfluidicchips formed with PDMS, using either dye. The PDMS may besurface-modified to minimize binding of these dyes to the chip.

Protocol C was foiled by the high background produced by dye binding toPDMS. After trapping a single cell in the chip, FM 1-43 bound to thechip more efficiently than to the membrane of the trapped cell.

Example 23 Capturing Cells in Single-Cell or Multi-Cell MicrofluidicChambers

This example describes capture of a single cell or a cell population ina microfluidic system; see FIGS. 80-82.

FIG. 80 shows a single cell captured at a retention site using a chipfabricated generally according to system 850 of Example 7. In FIG. 80A,cells 1610 follow a divided flow path extending in opposite directionsabove retention site 1612. In FIG. 80B, a trapped cell 1614 ispositioned at the retention site.

Multiple cells were captured in a larger retention chamber formed by achip fabricated generally according to system 250 of Example 2. FIGS.81A, 81B, and 81C show empty chamber 270, the chamber with two cells,and with six cells, respectively. FIG. 82 shows a similar capture ofcells, but here the cells are prelabeled with a fluorescent dye so thatthe cells are easily visible as bright green using fluorescentmicroscopy. FIGS. 82A and 82B show a chamber with only three cells andduring the entry of a fourth cell, respectively.

Example 24 Fixing and Staining Cells in a Microfluidic System

This example describes the use of a microfluidic system to fix a cellwith an organic solvent, methanol, and label the cell with acridineorange; see FIG. 83.

All cell manipulations and treatments were as described in Example 2.FIG. 83A shows a single cell 1630 retained at the bottom of retentionsite 1632. The cell is barely visible due to the low level of lightused. The cell was perfused with methanol to fix the cell, and visiblecell-shrinkage was evident (not shown). FIG. 83B shows that the cellexhibits no fluorescence. However, after the cell was perfused with asolution of acridine orange, the cell fluoresces brightly (see FIG.83C).

Example 25 Microfluidic Mechanism for Measuring Cell Secretion

This example describes the structure and use of a softlithography-based, microfluidic system for measuring secretion ofmolecules, complexes, and/or small particles from cells.

Many cell analyses measure release, and/or secretion of materials fromcells. In some cases, the cells secrete material naturally. For example,neurons are analyzed for their ability to secrete neurotransmitters atneural synapses; endocrine cells for secretion of endocrine hormones,such as insulin, growth hormone, prolactin, steroid hormones, etc.; anda broad range of cell types for secretion of cytokines. In other cases,cells are lysed to define an aspect of their internal contents. However,in any of these cases, a secreted or released material of interest mayno longer be held in a fixed position by the cells, and thus may be freeto diffuse into the ambient solution. Accordingly, such secreted orreleased materials may be difficult to analyze without concentratingthem and/or without using immobilized, high-affinity binding partners,for example, in ELISA.

Microfluidic systems may ameliorate some of the difficulties associatedwith measuring material released from cells, but may introduceadditional considerations. In microfluidic systems, cells may be grownin isolated chambers having small volumes, as described above in Example10. The chambers may maintain released materials in the small volumes,promoting subsequent analysis. However, to maintain the releasedmaterials in a concentrated form, the chambers may be isolated fromother portions of the microfluidic network. Such isolated chambers donot promote ready analysis of the released materials, since thematerials may be isolated from analytical reagents and may be difficultto collect without substantially diluting the released materials.Therefore, a microfluidic mechanism is needed that allows materialreleased from cells to be collected and/or analyzed in a distinctfluidic compartment that is not part of a primary fluidic layer of amicrofluidic system.

This example provides a microfluidic system having a cell chamber and aseparate material collection compartment that communicate fluidicallythrough a semi-permeable membrane. The semi-permeable membrane permitsmovement of material that is secreted/released from cells, but preventsmovement of cells themselves. The membrane may be form a portion of afluid layer, or interface with a fluid layer above and/or below thefluid layer. When disposed below, the membrane may form some or all ofthe substrate for the fluid layer. Accordingly, secreted/releasedmaterial may pass through the membrane for collection and/or analysis inanother compartment of the fluid layer, a compartment above the fluidlayer, and/or below the substrate. For example, the microfluidic systemmay include a layer similar to the base layer of Example 11.

Example 26 Microfluidic Analysis of a Heterogeneous ParticlePopulation—Part II

This example describes microfluidic systems for sorting and analyzingheterogeneous populations of particles, such as blood samples, based ondifferences in particle size; see FIGS. 84-88. Example 26 expands uponaspects of Example 15 above.

Description

This example provides a microfluidic system 1650 that selectivelyretains and analyzes larger particles from a mixture of larger andsmaller particles; see FIGS. 84 and 85. System 1650 includes an inputmechanism 1652, a positioning mechanism 1654, a filtration mechanism1656, a retention mechanism 1658, a perfusion mechanism 1660, a releasemechanism 1662, and a flow-based detection mechanism 1664, among others.These mechanisms may be grouped into a first set for inputting sampleand size-selecting the sample, and a second set for retaining, treating,measuring, and outputting the size-selected sample.

The first set of mechanisms may functionally interconnect as follows.Input mechanism 1652 introduces particles from a particle sample placedin particle input-reservoir 1666, into microfluidic network 1668 ofsystem 1650. Particles are moved by positioning mechanism 1654 tofiltration mechanism 1656 by flow along inlet channel 1670. Filtrationmechanism 1656 may act as a size-dependent and regulatable retentionmechanism, or prefilter, that removes smaller particles from theinputted particles, while retaining larger particles. After suitablefiltration, the larger particles may be released from filtrationmechanism 1656 and moved by positioning mechanism 1654 toward retentionmechanism 1658.

The second set of mechanisms may functionally interconnect as follows.Positioning mechanism 1654 may use a first focusing mechanism 1672 tofocus and direct particles toward retention mechanism 1658. Particlesretained by retention mechanism 1658 may be perfused with desiredreagents from perfusion mechanism 1660, then released by releasemechanism 1662. Released cells may be moved by positioning mechanism1654 toward flow-based detection mechanism 1664. During positioning,cells may be focused into a single stream of particles by a secondfocusing mechanism 1674. Finally, detected cells may be passed to outputmechanism 1676.

System 1650 may include a plurality of regulators, or valves, that mayregulate various aspects of the mechanisms described above; see FIG. 85.Valve V1 may regulate input mechanism 1652. Valve V2 may regulatealternative input mechanism 1678. Alternative input mechanism 1678 mayprovide an alternative source of input fluid, and may be used to supplyparticle-free fluid for washing filtration mechanism 1658, for carryingparticles from filtration mechanism to first focusing mechanism 1672 andon to retention mechanism 1658, and/or the like. Valve V3 may regulateinput from first reagent reservoir 1680. Valve V4 may regulate inputfrom second reagent reservoir 1682. Valve V5 may regulate flow of ashield buffer to space reagents from retained particles until thedesired moment for beginning treatment. V6 may regulate flow through afirst waste channel 1684. V7 may regulate release mechanism 1662. V8 mayregulate flow through a second waste channel 1686. V9 may regulate flowtoward detection mechanism 1664. Finally, V10 may regulate afilter-release mechanism 1688 that regulates release of particles fromregulatable retention mechanism 1656.

Further aspects of input mechanism 1652, positioning mechanism 1654,retention mechanism 1658, perfusion mechanism 1660, release mechanism1662, and output mechanism 1676 elsewhere in Section XIII.

Applications

The description that follows exemplifies use of system 1650 forseparation and analysis of white blood cells from a sample of wholeblood. However, system 1650 may be suitable for use with anyheterogeneous (or homogeneous) population of particles.

System 1650 first separates white blood cells from smaller red bloodcells and platelets. These separated white blood cells are directed to aretention site, retained, and then processed by the perfusion mechanismto stain the retained white blood cells. These stained cells are thenreleased from the retention site and then positioned to a separateflow-based detection site. The detection site then detects acharacteristic of the stained cells, based on the stainingmethod/reagents used.

A chip fabricated according to system 1650 may be readied for use asfollows. First, the chip may be loaded with water. Next, when all thechannels are filled, the water may be replaced with a buffer solution.At this point, the following valves generally are closed: V1, V2, V3,V4, V5, V9, and V10. By contrast, the following valves generally areopen: V6, V7, and V8. All input reservoirs may be loaded with theirrespective buffers/reagents. However, particle input-reservoir 1666typically is not loaded yet. Each waste reservoir 1692, 1694, 1696, and1698 may be emptied (or is already empty).

A sample of whole blood may be loaded and filtered as follows. Analiquot of blood is loaded into particle input-reservoir 1666. Valve V1may be opened and the blood allowed to flow into filtration mechanism1656. FIG. 86 shows the operation of filtration mechanism 1656 ingreater detail. A first set of particle-selective channels 1700, forexample, channels that are about 7 μm wide and 5 μm high, may bedisposed along the walls of inlet channel 1702. A second set ofparticle-selective channels or chamber channels 1704 also may bedisposed around the perimeter of capture chamber 1706. Accordingly, redblood cells may travel to flow-through chambers 1708 and then wastereservoirs 1692, 1694, along a substantial area formed by inlet channel1702 and chamber 1706. In particular, travel of red blood cells throughparticle-selective channels 1700 from inlet channel 1702 may avoidclogging chamber channels 1704. However, the white blood cells may beretained in chamber 1704, because they cannot pass through channels 1700and may not travel past chamber 1704 because filter-release mechanism1688 (valve V10) is closed.

White blood cells retained in capture chamber 1706 may be washed asfollows. After a suitable number of white blood cells have enteredchamber 1706, valve V1 may be closed so that no more whole blood entersinlet channel 1702 and chamber 1706. Then, valve V2 may be opened toallow the carrying buffer provided by alternative input mechanism 1678to wash residual red blood cells out of chamber 1706. At this point,waste reservoirs 1692, 1694 may be emptied to avoid reverse flow of thered blood cells back into chamber 1706.

Filtered white blood cells may be retained by retention mechanism 1658as follows; see FIGS. 85-87. Valve V10 may be opened to allow thefiltered white blood cells from chamber 1706 to be released. Thereleased cells may be focused by first focusing mechanism 1672 andcarried toward retention site 1710 (see FIG. 87). Flow of carryingbuffer from alternative input mechanism 1678 may act during this processto reposition the white blood cells from chamber 1706 to retention site1710.

Retained white blood cells may be stained with reagents as follows.Valve V10 may be closed to prevent additional white blood cells fromleaving chamber 1706 and entering retention site 1710. Next, valve V6may be closed to facilitate directing reagents along a flow path towardthe retained white blood cells by perfusion mechanism 1660. Next, whiteblood cells may be stained or otherwise treated/processed usingperfusion mechanism 1660, as described elsewhere in Section XIII,particularly Example 2. Pump P1 may be used by perfusion mechanism 1660to actively move reagents, buffer, and/or fluid during particletreatment (see FIG. 85). At this point, the valves may be in thefollowing configuration. Valves V1, V3, V4, V5, V6, V9, are V10 closed.Valves V2, V7, and V8 are open. After cell treatment has been completed,pump P1 may be turned off, and valves V3, V4, and V5 may be closed toterminate action of perfusion mechanism 1660.

Treated/processed cells may be released and detected as follows; seeFIGS. 84, 85, 87, and 88. Pump P2 may be turned on. This pump may beused to pull fluid, particles, and/or reaction products toward detectionmechanism 1664 and waste (output) reservoir 1698. Next, valve V8 may beclosed and valve V9 opened (see FIG. 87). With this valve configuration,fluid and particle may be directed toward waste reservoir 1698 insteadof waste reservoir 1696 (see FIG. 85). At this point, each focusingreservoir 1712, 1714, 1716, 1718 may be refilled with buffer and wastereservoir 1698 may be emptied. Then, partial or complete closure ofvalve V7 may be used to release white blood cells from retentionmechanism 1658. During release, buffer flowing from reservoirs 1712,1714, or alternative input mechanism 1678, may be used to carry thereleased white blood cells toward detection mechanism 1664. Bufferflowing from reservoirs 1716, 1718 may act in second focusing mechanism1674, to position (focus) the released cells to a desiredcross-sectional portion of outlet channel 1720, generally a centralportion (see FIG. 88). After cell focusing, outlet channel 1720 mayconstrict to a narrowed channel 1722, which may facilitate positioningthe cells in single file, that is, one-by-one at detection site 1724,rather than in groups.

System 1650 may be used to measure any suitable aspect of a blood sampleor other inputted particle population, including samples from patients,research subjects, volunteers, forensic studies, cadavers, etc. Suitableaspects may include analysis of leukemias, anemias, blood abnormalities,blood health, genetic diseases, infections, ratios of specific bloodcell types, presence of nonblood cells, and/or the like. Exemplaryleukemias may include acute lymphoblastic leukemias, chronic myelogenousleukemias, acute myelogenous leukemias, acute lymphoid leukemias,chronic lymphocystic leukemias, and/or juvenile myelolymphocysticleukemias, among others. Exemplary anemias and/or genetic diseases mayinclude aplastic anemias, Faconi anemias, sickle-cell anemias, and/orthe like. Other aspects or characteristics of blood cells (or otherheterogeneous particle populations) that may be suitable for analysisare described above in Sections VIII and XII.

FIG. 89 is a top plan view of a perfusion device for exposing particlesto an array of different reagents or different reagent concentrations.Here, microfluidic passage device 2000 provides a plurality ofgrowth/perfusion chambers 2030 for loading particles, such as cells,through loading passage 2010 which is controlled by valving line 2020which is in operable communication with control input 2070, and which,when actuated, isolates each chamber 2030 from one another. Particlesmay then be flushed out the chambers 2030 by opening valving line 2020and pushing fluid from loading passage 2010 through each chamber 2030towards exit passage 2080. Once each chamber 2030 is loaded withparticles, such as cells, and isolated, valve line 2040, which is inoperable communication with control input 2140, then opens to permitflow of reagent and diluent, such as media or a fluid that dilutes thereagent, through flow lines 2120, which originate from a diluentreservoir 2110, and optionally, reagent reservoir 2100, which may hold areagent for exposure to the particles. The ratio of diluent to reagentmay be controlled by valving, or, preferably, by controlling the bore ofthe lines connecting the diluent reservoir 2110 to flow line 2120 andreagent reservoir 2100 to flow line 2120. Diluent and reagent are thenfed into chambers 2030 by pumping action caused by, for example, aperistaltic pump 2090, which is actuated by pump input lines 2150 a-c,thus particles are perfused with reagent/diluent. Diluent, in the caseof cells, may be cell culture media. Effluent from chambers 2030 may becollected into waste reservoir 2050.

FIGS. 90 through 94 depict a top plan view of a device being used tomeasure the response of cells to a chemo-attractant. Microfluidicpassage device 2200 provides reagent loading chamber 2230, whereinreagent is metered into reagent chamber 2300 by the opening of valve2210 and blind filling reagent into reagent chamber 2300. Once reagentchamber 2230 is filled, particles 2320, such as cells, which werepreviously introduced into particle chamber 2300 are then exposed to agradient of reagent upon the opening of valve 2220, valve 2210,preferably, remains, closed during the formation of the gradient. FIG.91 shows reagent entering into gradient forming mechanism 2250, whichhas channels 2270 for limiting reagent flow into particle chamber 2320.FIG. 92 depicts the advancement of reagent towards particle chamber2300. FIGS. 93 and 94 depict the movement of particles 2320 towardchannels 2270 where the chemo-attractant reagent is emanating from.

2230 is filled, particles 2320, such as cells, which were previouslyintroduced into particle chamber 2300 are then exposed to a gradient ofreagent upon the opening of valve 2220, valve 2210, preferably, remainsclosed during the formation of the gradient. FIG. 91 shows reagententering into gradient forming mechanism 2250, which has channels 2270for limiting reagent flow into particle chamber 2320. FIG. 92 depictsthe advancement of reagent towards particle chamber 2300. FIGS. 93 and94 depict the movement of particles 2320 toward channels 2270 where thechemo-attractant reagent is emanating from.

FIG. 95 is a close-up top plan view of a perfusion chamber withassociated valving system. Particles, such as cells, can be loaded intoa series of particle chambers 2450 by opening isolation valve line 2430which, when closed, isolates each chamber 2450 from each other.Particles do not enter flow line 2460 since they are retained in chamber2450 by screen or comb 2490, which each obstruction is spaced-apart fromthe other at a distance less than that of the particle, so as to retainthe particle on one side of the screen or comb 2490. In use, particlesare introduced into chamber 2450 by the opening of isolation valve line2430 which allows the particles to flow through and fill each chamber2450. Once filled with the desired amount of particles, isolation valves2430 are closed to isolate each chamber 2450 from each other, and thenflow valves 2440 are opened to allow for flow of reagent through chamber2450 to perfuse the particles with reagent. Once an experiment iscomplete, flow valves 2440 may then be closed, isolation valves 2430 maythan be opened to flush out particles. If the particles are adherentcells, such cells can be liberated if attached by exposing such adheredcells to a cell dislodging reagent such as trypsin. Once liberated, thecells can be flushed out of the system, and the system reused.

FIGS. 96 a through 96 c depict a preferred embodiment of a perfusionchamber device wherein a plurality of different compounds from aplurality of different compound sources can be perfused through aplurality of cell chambers such that each different compound is perfusedthrough a different chamber, and then a common detection reagent can beperfused through each different cell chamber from a common detectionreagent source. In some embodiments, the different compounds areperfused in one direction through each cell chamber and the commondetection agent is perfused in an different direction through thedetection chamber. In some embodiments, the common detection reagentsource is in fluid communication, preferably selectively, with a wastereceptacle. In some embodiments, the different compound sources may alsoserve as a second waste receptacle after the different compounds havebeen delivered to the different cell chambers.

For example, FIG. 96 a shows a device 2400 wherein cells are introducedthrough cell inlet 2480 and flowed through until line 2410 is filledwith cell containing solution. During cell loading, chamber valves 2440are closed, and, preferably, detection reagent valve 2448 and wastevalve 2447 remain closed. Once chambers 2450 have been loaded withcells, chamber isolation valves 2430 close to isolate each chamber fromthe other, as shown in FIG. 96 b. As shown in FIG. 96 c, with isolationvalves 2430 remaining closed, cells contained within chambers 2450 arethen exposed different compounds supplied by opening valves 2440, whichpermits flow of the different compounds through channels 2460 and 2500into chambers 2450, perfusing across through chamber 2450, and outthrough to waste 2441. After a selected period of perfusion, waste valve2447 may be closed and detection reagent valve 2448 may be openedwherein a reagents, for example, media, wash solution, detectionagent(s), and/or developing reagents may be flowed back through chambers2450 out through channels 2460 and 2500. Alternatively, a single commoncompound may be first flowed to each of chambers 2450 from detectionreagent input 2448, and then different detection reagents may be flowedthrough each chamber from channels 2460 and 2500.

Perfusion type devices, such as those described above, may be useful forconducting toxicological assays. The invention provides in one aspectfor devices and methods for conducting cell toxicity assays whereincells are exposed, preferably transiently for a selected period of time,to a compound which may be toxic to the cells, or becomes toxic to thecells through further processing. For example, liver cells, previouslyloaded into chambers, may be introduced into the chambers, and then adrug candidate, or a plurality of drug candidates may be presented tothe cells within the chambers for a selected period of time and/or atselected concentrations. The drug candidates may then be flushed out bya wash step, which is then followed, after a selected period of time, bya detection treatment, where the drug exposed cells are treated with areagent to detect a change in state caused by the drug candidate. Insome embodiments, no detection reagent is used. Instead, a change insome physical property of the cells is observed or measured, such asimpedance, resistivity, conductivity, cell morphology, proliferation,and lysis. Changes in state may include, for example, apoptosis,proliferation, senescence, changes in membrane chemistry, and changes innuclear or organelle structure. Detection reagents may include, annexinV type assays, apoptosis detection reagents, vital dye reagents, Quinn2dyes, LDH Assays: (lactase dehydrogenase enzyme leakage from plasmamembrane), ATP measurements (cell proliferation/cytotocicityassessment), and MTT salt assay, WST-1 type assays.

Cell assays may include: trypan blue; eosin Y nigrosine; propidiumiodide; ethidium bromide, wherein dead and viable cells arediscriminated by differential staining and counted using a light orfluorescence microscope. These methods do not allow the processing oflarge sample numbers and do not account for dead cells which may havelysed. Thus, the rate of cell death in long term cultures can beunderestimated. In other embodiments, fluorescent dyes: [51Cr];[3H]-thymidine; [3H]-proline; [75Se]-methionine;[125J]-5-iodo-2-deoxyuridine; bis-carboxyethyl-carboxyfluorescein(BCECF); calcein-AM from prelabeled target cells.

Yet another embodiment includes assays based on the measurement ofcytoplasmic enzyme activity released by damaged cells. The amount ofenzyme activity detected in the culture supernatant correlates to theproportion of lysed cells. Enzyme release assays have been described foralkaline and acid phosphatase; glutamate-oxalacetate transaminase;glutamate pyruvate transaminase; arginosuccinate lyase.

The disclosure set forth above may encompass one or more distinctinventions, with independent utility. Each of these inventions has beendisclosed in its preferred form(s). These preferred forms, including thespecific embodiments thereof as disclosed and illustrated herein, arenot intended to be considered in a limiting sense, because numerousvariations are possible. The subject matter of the inventions includesall novel and nonobvious combinations and subcombinations of the variouselements, features, functions, and/or properties disclosed herein.

1. A microfluidic device for exposing cells to a plurality of compoundscomprising: (a) a cell inlet; (b) a plurality of cell chambers, each influidic communication with each other and said cell inlet; (c) aplurality of chamber isolation valves, said isolation valves, whenactuated, fluidically isolating said chambers from each other; (d) aplurality of compound inlets and outlets, each compound inlet and outletbeing in fluid communication with one of said chambers, said compoundinlets and outlets further comprising a cell retention sieve forretaining cells within said chambers while permitting fluid flow throughsaid chambers so that said cells are perfused with said fluid when saidfluid is flowed through said chambers from said compound inlets towardssaid compound outlets, said compound inlets being selectively in fluidcommunication with a plurality of compound sources, and said compoundoutlet being in fluid communication selectively either with a commonwaste receptacle or a common reagent reservoir; (e) a plurality ofcompound valves in communication with said compound inlets and outlets,said valves being capable fluidically isolating said chamber from saidcompound sources and said common receptacle and common reagentreservoir; (f) a common waste receptacle valve, said common wastereceptacle valve being capable of fluidically isolating said commonwaste receptacle from said chamber when actuated; and, (g) a commonreagent reservoir valve, said common reagent reservoir valve beingcapable of fluidically isolating said common reagent receptacle fromsaid chamber when actuated.
 2. The device of claim 1 wherein at leastone of said plurality of compounds is a cell toxic compound.
 3. Thedevice of claim 1 where said common reagent reservoir contains adetection agent responsive to a toxic response of said cells to one ofsaid compounds.
 4. A method for determining whether one of a pluralityof compounds causes a toxic response in one of a plurality of cellscomprising the steps of: providing the device of claim 1; introducingsaid cells into said device of claim 1, isolating said cells within saidchambers of said device of claim 1, exposing said cells to saidplurality of said compounds while said cells are retained in saidchambers, determining whether any of said cells had a toxic responseresulting from exposure to one of said plurality of compounds.